Messenger RNA therapy for the treatment of Friedreich&#39;s ataxia

ABSTRACT

The present invention provides methods and compositions of treating Friedreich&#39;s ataxia (FRDA) based on administering an mRNA encoding a frataxin protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication Ser. No. 62/521,734, filed Jun. 19, 2017, the disclosure ofwhich is hereby incorporated by reference in its entirety for allpurposes.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety for all purposes. Said ASCII copy, created on Jun. 18,2018, is named MRT-2004US_SL.txt and is 18,135 bytes in size.

BACKGROUND

Friedreich's ataxia (also called FA or FRDA) is a rare inherited diseasethat causes nervous system damage and movement problems. It usuallybegins in childhood and leads to impaired muscle coordination (ataxia)that worsens over time. In Friedreich's ataxia, the spinal cord,peripheral nerves and cerebellum degenerate. This damage results inawkward, unsteady movements and impaired sensory functions. The disorderalso causes problems in the heart and spine, and some people with thecondition develop diabetes.

Friedreich's ataxia is caused by a defect (mutation) in the FXN genethat encodes the protein frataxin. This protein is found in cellsthroughout the body, with the highest levels in the heart, spinal cord,liver, pancreas, and muscles used for voluntary movement (skeletalmuscles). Within cells, frataxin is found in energy-producing structurescalled mitochondria. Although its function is not fully understood,frataxin appears to help assemble clusters of iron and sulfur moleculesthat are critical for the function of many proteins, including thoseneeded for energy production. One region of the FXN gene contains asegment of DNA known as a GAA trinucleotide repeat. In most people, thenumber of GAA repeats in the FXN gene is fewer than 12 (referred to asshort normal). Sometimes, however, the GAA segment is repeated 12 to 33times (referred to as long normal).

Friedreich's ataxia results from an increased number of copies(expansion) of the GAA trinucleotide repeat in the FXN gene. In peoplewith this condition, the GAA segment is abnormally repeated 66 to morethan 1,000 times. The length of the GAA trinucleotide repeat appears tobe related to the age at which the symptoms of Friedreich's ataxiaappear. People with GAA segments repeated fewer than 300 times tend tohave a later appearance of symptoms (after age 25) than those withlarger GAA trinucleotide repeats.

Most individuals with Friedreich's ataxia have the expanded GAAtrinucleotide repeat in both copies of the FXN gene. It is not fullyunderstood how FXN gene mutations cause Friedreich's ataxia. Mutationsin this gene disrupt production of frataxin, greatly reducing the amountof this protein in cells. A shortage of frataxin appears to decrease theactivity of proteins that contain iron-sulfur clusters, which couldimpair the production of energy in mitochondria. Cells with insufficientamounts of frataxin are also particularly sensitive to reactivemolecules (free radicals) that can damage and destroy cells. Cells inthe brain, spinal cord, and muscles that are damaged or have inadequateenergy supplies may not function properly, leading to the signs andsymptoms of Friedreich's ataxia.

As with many degenerative diseases of the nervous system, there iscurrently no cure or effective treatment for Friedreich's ataxia.However, many of the symptoms and accompanying complications can betreated to help individuals maintain optimal functioning as long aspossible. Doctors can prescribe treatments for diabetes, if present;some of the heart problems can be treated with medication as well.Orthopedic problems such as foot deformities and scoliosis can becorrected with braces or surgery, while physical therapy may prolong useof the arms and legs.

SUMMARY OF THE INVENTION

The present invention provides, among other things, improved methods andcompositions for the treatment of Friedreich's ataxia (FRDA) based onmRNA therapy. The invention encompasses the observation thatadministration of an mRNA encoding a human frataxin (FXN) protein,encapsulated within a liposome, resulted in highly efficient andsustained protein production in vivo.

In one aspect, the present invention provides methods of treatingFriedreich's ataxia (FRDA), comprising administering to a subject inneed of treatment a composition comprising an mRNA encoding a frataxinprotein.

In another aspect, the present invention generally provides methods ofdelivering frataxin in vivo, comprising administering periodically to asubject in need of delivery a composition comprising an mRNA encoding afrataxin protein such that the frataxin protein is expressed in vivo ata level sufficient to achieve therapeutic efficacy (e.g., a level of atleast 10% of a normal control level).

In still another aspect, the present invention provides pharmaceuticalcompositions for treating Friedreich's ataxia (FRDA) containing an mRNAencoding a frataxin protein.

In various embodiments according to the invention, an mRNA encoding afrataxin protein comprises a polynucleotide sequence at least 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, an mRNA encodinga frataxin protein comprises a polynucleotide sequence at least 80%identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.In certain embodiments, an mRNA encoding a frataxin protein comprises apolynucleotide sequence at least 90% identical to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, an mRNAencoding a frataxin protein comprises a polynucleotide sequence at least95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:4. In certain embodiments, an mRNA encoding a frataxin protein comprisesa polynucleotide sequence identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, or SEQ ID NO: 4.

In various embodiments, an mRNA encoding a frataxin protein is codonoptimized. In some embodiments, the mRNA comprises a polynucleotidesequence wherein the percentage of guanine nucleotides is at least 28%,the percentage of cytosine nucleotides is at least 30%, the combinedpercentage of guanine and cytosine nucleotides is at least 59%, and/orthe percentage of adenine nucleotides is no more than 22.5%. In someembodiments, the mRNA comprises a polynucleotide sequence wherein thepercent change in the number of guanine nucleotides relative to a nativehuman FXN mRNA sequence is at least is at least 9%, the percent changein the number of cytosine nucleotides relative to a native human FXNmRNA sequence is at least is at least 7%, the percent change in thecombined number of guanine and cytosine nucleotides relative to a nativehuman FXN mRNA is at least 9%, and/or the percent change in the numberof adenine nucleotides relative to a native human FXN mRNA sequence isat least −7%.

In some embodiments, an mRNA further comprises a 5′ untranslated region(UTR) and/or a 3′ untranslated region (UTR). In some embodiments, the 5′UTR includes a sequence of SEQ ID NO: 6. In some embodiments, the 3′ UTRincludes a sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

In some embodiments, the composition is administered to the subject viaintravenous (IV) delivery. In some embodiments, the composition isadministered to the subject via intrathecal (IT) delivery.

Typically, an mRNA encoding a frataxin protein is administeredperiodically (e.g., once a day, twice a week, once a week, once everyother week, twice a month, once every 14 days, once a month, etc.). Insome embodiments, the mRNA is administered once a week. In someembodiments, the mRNA is administered twice a month. In someembodiments, the mRNA is administered once every 14 days. In someembodiments, the mRNA is administered once a month.

In some embodiments, administration of the composition results inexpression of the frataxin protein in various tissues in vivo. In someembodiments, administration of the composition results in expression ofthe frataxin protein detectable in the liver, the spinal cord, thebrain, the cerebellum, and/or at least one dorsal root ganglion (DRG) ofthe subject. In some embodiments, the frataxin protein is detectable atleast 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2 weeksafter administering the composition. In some embodiments, the frataxinprotein is detectable at a level of at least 0.1 pg/μg of total protein,at least 0.5 pg/μg of total protein, at least 1.0 pg/μg of totalprotein, at least 5 pg/μg of total protein, at least 10 pg/μg of totalprotein, at least 50 pg/μg of total protein, or at least 100 pg/μg oftotal protein. In some embodiments, the frataxin protein is detectableat a level of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% ofa normal control level. In some embodiments, the frataxin protein isdetectable at a level of at least 10%, 20%, 30%, 40%, 50%, 1-fold,1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5-foldgreater than a control level indicative of untreated patient.

In some embodiments, the mRNA is administered at a dose of at least 1μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, or 20μg.

In various embodiments, the mRNA is encapsulated within a liposome. Insome embodiments, the liposome comprises one or more cationic lipids,one or more non-cationic lipids, and one or more PEG-modified lipids.

In some embodiments, the one or more cationic lipids comprise a cationiclipid selected from the group consisting of C12-200, MC3, DLinDMA,DLinkC2DMA, cKK-E12, ICE (Imidazole-based), HGT5000, HGT5001 (CCBene),OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC,DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP,DLincarbDAP, DLinCDAP, DLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, andcombinations thereof.

In some embodiments, the one or more non-cationic lipids comprise anon-cationic lipid selected from the group consisting of DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) and combinationsthereof.

In some embodiments, the liposome further comprises one or morecholesterol-based lipids. For example, the one or more cholesterol-basedlipids may be cholesterol and/or PEGylated cholesterol.

In some embodiments, the liposome further comprises a sphingomyelin. Forexample, the sphingomyelin may be a brain sphingomyelin.

In some embodiments, the mRNA comprises one or more modifiednucleotides. For example, the one or more modified nucleotides may bepseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine,2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine.

In some embodiments, the mRNA is unmodified.

It is to be understood that all embodiments as described above areapplicable to all aspects of the present invention. Other features,objects, and advantages of the present invention are apparent in thedetailed description, drawings, and claims that follow. It should beunderstood, however, that the detailed description, the drawings, andthe claims, while indicating embodiments of the present invention, aregiven by way of illustration only, not limitation. Various changes andmodifications within the scope of the invention will be apparent tothose skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING

The drawings are for illustration purposes only not for limitation.

FIG. 1 depicts exemplary detection of human frataxin protein by Westernblot after HEK cells were transfected with hFXN mRNA-loaded liposomes.

FIG. 2 depicts exemplary detection of human frataxin protein by Westernblot in liver samples from mice treated with a single intravenous doseof hFXN mRNA-loaded liposomes.

FIG. 3A depicts exemplary detection of human frataxin protein by ELISAin liver samples from mice treated with a single intravenous dose ofhFXN mRNA-loaded liposomes.

FIG. 3B depicts exemplary detection of hFXN mRNA by RT qPCR in liversamples from mice treated with a single intravenous dose of hFXNmRNA-loaded liposomes.

FIG. 4A depicts exemplary detection of human frataxin protein by ELISAin heart samples from mice treated with a single intravenous dose ofhFXN mRNA-loaded liposomes.

FIG. 4B depicts exemplary detection of human frataxin protein by ELISAin DRG samples from mice treated with a single intravenous dose of hFXNmRNA-loaded liposomes.

FIG. 5A depicts exemplary detection of human frataxin protein by Westernblot in DRG samples from mice treated with a single intrathecal dose ofhFXN mRNA-loaded liposomes.

FIG. 5B depicts exemplary detection of human frataxin protein by Westernblot in spinal cord samples from mice treated with a single intrathecaldose of hFXN mRNA-loaded liposomes.

FIG. 6 depicts exemplary detection of human frataxin protein by Westernblot in liver samples from mice treated with a single intrathecal doseof hFXN mRNA-loaded liposomes.

FIG. 7 depicts exemplary detection of human frataxin protein by ELISA inliver samples from mice treated with a single intrathecal dose of hFXNmRNA-loaded liposomes.

FIG. 8A depicts exemplary detection of human frataxin protein by ELISAin DRG samples from mice treated with a single intrathecal dose of hFXNmRNA-loaded liposomes.

FIG. 8B depicts exemplary detection of human frataxin protein by ELISAin spinal cord samples from mice treated with a single intrathecal doseof hFXN mRNA-loaded liposomes.

FIG. 9A depicts exemplary detection of human frataxin protein by ELISAin cerebellum samples from mice treated with a single intrathecal doseof hFXN mRNA-loaded liposomes.

FIG. 9B depicts exemplary detection of human frataxin protein by ELISAin cerebrum samples from mice treated with a single intrathecal dose ofhFXN mRNA-loaded liposomes.

FIG. 10A depicts exemplary detection of human frataxin protein by ELISAin DRG samples from mice treated with a single intrathecal dose of hFXNmRNA-loaded liposomes.

FIG. 10B depicts exemplary detection of human frataxin protein by ELISAin spinal cord samples from mice treated with a single intrathecal doseof hFXN mRNA-loaded liposomes.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference for all purposes.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active.

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinis expressed and retained within the target tissue (also referred to as“local distribution” or “local delivery”), and situations in which anmRNA is delivered to a target tissue and the encoded protein isexpressed and secreted into patient's circulation system (e.g., serum)and systematically distributed and taken up by other tissues (alsoreferred to as “systemic distribution” or “systemic delivery).

Encapsulation: As used herein, the term “encapsulation,” or grammaticalequivalent, refers to the process of incorporating an mRNA molecule intoa nanoparticle.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a polypeptide, assemble multiplepolypeptides (e.g., heavy chain or light chain of antibody) into anintact protein (e.g., antibody) and/or post-translational modificationof a polypeptide or fully assembled protein (e.g., antibody). In thisapplication, the terms “expression” and “production,” and grammaticalequivalents, are used interchangeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required fora quantity such as nucleic acid or protein concentration or activity tofall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide. mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions. mRNA can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, mRNA can comprisenucleoside analogs such as analogs having chemically modified bases orsugars, backbone modifications, etc. An mRNA sequence is presented inthe 5′ to 3′ direction unless otherwise indicated.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA. Furthermore, theterms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleicacid analogs, i.e., analogs having other than a phosphodiester backbone.For example, the so-called “peptide nucleic acids,” which are known inthe art and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. Theterm “nucleotide sequence encoding an amino acid sequence” includes allnucleotide sequences that are degenerate versions of each other and/orencode the same amino acid sequence. Nucleotide sequences that encodeproteins and/or RNA may include introns. Nucleic acids can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated. In someembodiments, a nucleic acid is or comprises natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and2-thiocytidine); chemically modified bases; biologically modified bases(e.g., methylated bases); intercalated bases; modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/ormodified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages). In some embodiments, the presentinvention is specifically directed to “unmodified nucleic acids,”meaning nucleic acids (e.g., polynucleotides and residues, includingnucleotides and/or nucleosides) that have not been chemically modifiedin order to facilitate or achieve delivery.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre- and post-natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, methods ofdelivering compositions comprising mRNA encoding a frataxin protein andmethods and compositions for treating Friedreich's ataxia (FRDA) basedon mRNA therapy. In particular, the present invention provides methodsof delivering a composition in vivo, the method comprising administeringthe composition to a subject, wherein the composition comprises an mRNAencoding a frataxin protein, and wherein the mRNA is encapsulated withina liposome. The present invention further provides methods of deliveringa composition in vivo, the method comprising administering thecomposition to a subject, wherein the composition comprises an mRNAencoding a frataxin protein, and wherein the mRNA comprises apolynucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the mRNA isencapsulated within one or more liposomes. As used herein, the term“liposome” refers to any lamellar, multilamellar, or solid nanoparticlevesicle. Typically, a liposome as used herein can be formed by mixingone or more lipids or by mixing one or more lipids and polymer(s). Thus,the term “liposome” as used herein encompasses both lipid and polymerbased nanoparticles. In some embodiments, a liposome suitable for thepresent invention contains one or more of cationic lipid(s),non-cationic lipid(s), cholesterol-based lipid(s), PEG-modifiedlipid(s), and a sphingomyelin.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Friedreich's Ataxia (FRDA)

The present invention may be used to treat a subject who is sufferingfrom or susceptible to FRDA (i.e., a subject in need of treatment). FRDAis a genetic disorder characterized by mutations in the gene forfrataxin (FXN). Within cells, frataxin is found in energy-producingstructures called mitochondria. Frataxin appears to help assembleclusters of iron and sulfur molecules that are critical for the functionof many proteins, including those needed for energy production.

Symptoms of FRDA include: reduced frataxin levels (e.g., reducedfunctional frataxin levels), weight loss, early mortality (median age ofdeath, 35 years), defects in motor coordination, defects in musclestrength, gait ataxia, and scoliosis. Characteristics of FRDA pathologyinclude: degeneration of the dorsal root ganglia (DRG), retinal celldegeneration, increased ferritin levels, increased ferroportin levels,iron accumulation, cardiac fibrosis, cardiomyopathy, aconitase activity,activation of apoptosis and autophagy and reduced myelin sheaththickness.

Frataxin (FXN)

In some embodiments, the present invention provides methods andcompositions for delivering mRNA encoding FXN to a subject for thetreatment of FRDA. A suitable FXN mRNA encodes any full length, fragmentor portion of a frataxin protein which can be substituted fornaturally-occurring frataxin protein activity and/or reduce theintensity, severity, and/or frequency of one or more symptoms associatedwith FRDA.

In some embodiments, a suitable mRNA sequence is a human mRNA sequence(hFXN) encoding a human frataxin protein. In some embodiments, asuitable mRNA sequence is a codon-optimized mRNA sequence encoding ahuman frataxin protein.

Codon-optimized human FXN mRNA coding sequence #1 (SEQ ID NO: 1)AUGUGGACCCUGGGUCGGAGAGCUGUGGCCGGUCUGCUGGCUUCCCCCUCACCGGCACAAGCGCAGACCCUGACUAGAGUGCCUAGGCCCGCUGAACUCGCACCACUGUGCGGCAGACGGGGACUCCGGACUGACAUCGAUGCCACCUGUACCCCGCGAAGGGCAUCUAGCAAUCAGCGCGGACUGAACCAGAUCUGGAACGUGAAGAAGCAGUCCGUGUACCUGAUGAAUCUGCGCAAAUCCGGCACUCUCGGACACCCGGGAUCGCUGGAUGAGACUACUUACGAGCGCUUGGCCGAAGAAACCCUGGAUUCGCUGGCCGAGUUUUUCGAGGACCUGGCCGACAAGCCCUACACGUUCGAGGACUACGACGUGUCCUUCGGAUCGGGCGUGCUGACCGUGAAGCUCGGCGGGGAUUUGGGGACCUACGUGAUCAACAAGCAGACACCGAACAAGCAAAUUUGGCUCUCCUCCCCUUCCUCCGGACCUAAGCGCUACGACUGGACCGGGAAGAACUGGGUCUACUCCCAUGACGGCGUCAGCCUUCACGAACUGCUGGCCGCCGAACUGACUAAGGCCCUCAAAACUAAGCUGGACCUGUCGAGCCUUGCCUAUUCCGGAAAGGACGCCUGACodon-optimized human FXN mRNA coding sequence #2 (SEQ ID NO: 2)AUGUGGACCCUGGGACGCAGAGCCGUGGCUGGCCUUCUGGCCUCCCCAAGCCCUGCCCAAGCCCAGACCUUGACUAGAGUGCCUAGACCGGCCGAACUCGCUCCCCUGUGCGGACGGAGGGGACUCAGGACUGACAUCGACGCAACAUGCACUCCAAGACGCGCCUCCAGCAACCAGCGGGGCCUCAACCAGAUUUGGAACGUGAAAAAGCAGUCCGUCUAUCUGAUGAACCUCCGCAAGUCCGGCACCUUGGGGCAUCCCGGGUCACUGGAUGAAACCACCUACGAACGGCUGGCCGAAGAGACUCUCGACUCCCUGGCCGAGUUCUUCGAGGACCUGGCGGAUAAGCCGUACACUUUCGAGGACUACGAUGUCUCUUUCGGAUCCGGCGUGCUGACCGUGAAGCUCGGUGGCGACCUCGGAACUUACGUGAUCAACAAGCAAACGCCCAACAAGCAGAUCUGGCUGUCCUCGCCGUCAUCGGGACCUAAGCGCUACGAUUGGACCGGGAAGAAUUGGGUGUACUCGCACGACGGUGUCAGCCUGCACGAGCUGCUUGCGGCGGAACUGACCAAGGCACUCAAGACCAAACUGGACCUGUCCAGCCUGGCCUACUCCGGAAAGGACGCCUAGCodon-optimized human FXN mRNA coding sequence #3 (SEQ ID NO: 3)AUGUGGACUCUGGGCCGGAGAGCUGUGGCAGGCCUUCUCGCCUCGCCAUCCCCUGCCCAAGCGCAGACCCUGACUAGGGUCCCUAGGCCUGCCGAGUUGGCACCGUUGUGCGGUCGGAGAGGACUGCGCACCGACAUCGAUGCCACCUGUACUCCUCGGAGAGCCUCGUCCAACCAGCGGGGCCUGAACCAGAUCUGGAACGUGAAGAAACAGUCCGUCUACCUGAUGAACCUCCGCAAGUCGGGAACCCUGGGACAUCCGGGUUCCCUGGAUGAGACUACGUACGAACGGCUGGCGGAAGAAACCCUGGACUCCCUGGCGGAGUUCUUCGAGGACCUGGCUGACAAGCCCUACACUUUUGAGGACUACGACGUGUCAUUCGGAAGCGGAGUGUUGACAGUGAAGCUGGGGGGCGAUCUGGGAACCUACGUGAUCAACAAGCAGACCCCGAACAAGCAAAUUUGGCUGUCCUCACCCUCCUCCGGACCUAAACGCUACGACUGGACCGGGAAGAACUGGGUGUAUAGCCACGACGGUGUCAGCCUUCACGAACUGCUUGCGGCCGAACUGACCAAGGCCCUCAAGACCAAGCUCGAUCUGUCUAGCCUCGCCUACUCCGGAAAGGACGCCUGACodon-optimized human FXN mRNA coding sequence #4 (SEQ ID NO: 4)AUGUGGACCUUGGGACGGAGAGCCGUGGCUGGACUGUUGGCCUCUCCUUCCCCGGCACAAGCCCAAACUCUGACCCGGGUCCCUAGACCGGCAGAGCUGGCUCCCCUGUGUGGUCGGCGGGGACUGAGAACUGAUAUUGACGCCACAUGCACUCCUAGGCGCGCGAGCUCCAAUCAGCGCGGCCUGAACCAGAUCUGGAACGUGAAGAAGCAGUCCGUCUACUUGAUGAACCUCCGCAAGUCCGGCACUCUGGGCCAUCCGGGAUCCCUCGACGAGACUACCUACGAGCGGCUGGCGGAAGAAACCCUGGAUUCCCUGGCCGAAUUCUUCGAGGACCUGGCCGACAAGCCCUACACCUUUGAGGACUACGACGUGUCCUUCGGAUCGGGAGUGCUGACCGUGAAGCUCGGCGGAGAUCUCGGGACUUAUGUGAUCAACAAGCAGACGCCGAACAAGCAGAUCUGGCUUAGCUCACCCUCGAGCGGACCAAAGCGCUACGACUGGACCGGCAAAAACUGGGUGUACUCCCACGAUGGUGUCAGCCUUCACGAACUGCUGGCCGCGGAACUGACCAAGGCCCUUAAGACCAAGCUCGACCUCUCAUCCCUGGCCUACUCCGGGAAAGACGCGUGA Human FXN protein sequence (SEQ ID NO: 5)MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDAExemplary Codon-Optimized Frataxin (FXN) mRNAs

Construct Design:

-   X—SEQ ID NO: 1—Y-   X—SEQ ID NO: 2—Y-   X—SEQ ID NO: 3—Y-   X—SEQ ID NO: 4—Y

5′ and 3′ UTR Sequences:

(SEQ ID NO: 6) X (5′ UTR Sequence) = GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACC GUCCUUGACACG(SEQ ID NO: 7) Y (3′ UTR Sequence) = CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU OR (SEQ ID NO: 8)GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCA AAGCU

In one embodiment, a codon-optimized human FXN mRNA sequence includesSEQ ID NO: 1.

In one embodiment, a full-length codon-optimized human FXN mRNA sequenceis:

(SEQ ID NO: 9) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACCCUGGGUCGGAGAGCUGUGGCCGGUCUGCUGGCUUCCCCCUCACCGGCACAAGCGCAGACCCUGACUAGAGUGCCUAGGCCCGCUGAACUCGCACCACUGUGCGGCAGACGGGGACUCCGGACUGACAUCGAUGCCACCUGUACCCCGCGAAGGGCAUCUAGCAAUCAGCGCGGACUGAACCAGAUCUGGAACGUGAAGAAGCAGUCCGUGUACCUGAUGAAUCUGCGCAAAUCCGGCACUCUCGGACACCCGGGAUCGCUGGAUGAGACUACUUACGAGCGCUUGGCCGAAGAAACCCUGGAUUCGCUGGCCGAGUUUUUCGAGGACCUGGCCGACAAGCCCUACACGUUCGAGGACUACGACGUGUCCUUCGGAUCGGGCGUGCUGACCGUGAAGCUCGGCGGGGAUUUGGGGACCUACGUGAUCAACAAGCAGACACCGAACAAGCAAAUUUGGCUCUCCUCCCCUUCCUCCGGACCUAAGCGCUACGACUGGACCGGGAAGAACUGGGUCUACUCCCAUGACGGCGUCAGCCUUCACGAACUGCUGGCCGCCGAACUGACUAAGGCCCUCAAAACUAAGCUGGACCUGUCGAGCCUUGCCUAUUCCGGAAAGGACGCCUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

In one embodiment, another full-length codon-optimized human FXN mRNAsequence is:

(SEQ ID NO: 10) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACCCUGGGUCGGAGAGCUGUGGCCGGUCUGCUGGCUUCCCCCUCACCGGCACAAGCGCAGACCCUGACUAGAGUGCCUAGGCCCGCUGAACUCGCACCACUGUGCGGCAGACGGGGACUCCGGACUGACAUCGAUGCCACCUGUACCCCGCGAAGGGCAUCUAGCAAUCAGCGCGGACUGAACCAGAUCUGGAACGUGAAGAAGCAGUCCGUGUACCUGAUGAAUCUGCGCAAAUCCGGCACUCUCGGACACCCGGGAUCGCUGGAUGAGACUACUUACGAGCGCUUGGCCGAAGAAACCCUGGAUUCGCUGGCCGAGUUUUUCGAGGACCUGGCCGACAAGCCCUACACGUUCGAGGACUACGACGUGUCCUUCGGAUCGGGCGUGCUGACCGUGAAGCUCGGCGGGGAUUUGGGGACCUACGUGAUCAACAAGCAGACACCGAACAAGCAAAUUUGGCUCUCCUCCCCUUCCUCCGGACCUAAGCGCUACGACUGGACCGGGAAGAACUGGGUCUACUCCCAUGACGGCGUCAGCCUUCACGAACUGCUGGCCGCCGAACUGACUAAGGCCCUCAAAACUAAGCUGGACCUGUCGAGCCUUGCCUAUUCCGGAAAGGACGCCUGAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU

In one embodiment, a codon-optimized human FXN mRNA sequence includesSEQ ID NO: 2.

In one embodiment, a full-length codon-optimized human FXN mRNA sequenceis:

(SEQ ID NO: 11) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACCCUGGGACGCAGAGCCGUGGCUGGCCUUCUGGCCUCCCCAAGCCCUGCCCAAGCCCAGACCUUGACUAGAGUGCCUAGACCGGCCGAACUCGCUCCCCUGUGCGGACGGAGGGGACUCAGGACUGACAUCGACGCAACAUGCACUCCAAGACGCGCCUCCAGCAACCAGCGGGGCCUCAACCAGAUUUGGAACGUGAAAAAGCAGUCCGUCUAUCUGAUGAACCUCCGCAAGUCCGGCACCUUGGGGCAUCCCGGGUCACUGGAUGAAACCACCUACGAACGGCUGGCCGAAGAGACUCUCGACUCCCUGGCCGAGUUCUUCGAGGACCUGGCGGAUAAGCCGUACACUUUCGAGGACUACGAUGUCUCUUUCGGAUCCGGCGUGCUGACCGUGAAGCUCGGUGGCGACCUCGGAACUUACGUGAUCAACAAGCAAACGCCCAACAAGCAGAUCUGGCUGUCCUCGCCGUCAUCGGGACCUAAGCGCUACGAUUGGACCGGGAAGAAUUGGGUGUACUCGCACGACGGUGUCAGCCUGCACGAGCUGCUUGCGGCGGAACUGACCAAGGCACUCAAGACCAAACUGGACCUGUCCAGCCUGGCCUACUCCGGAAAGGACGCCUAGCGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

In one embodiment, another full-length codon-optimized human FXN mRNAsequence is:

(SEQ ID NO: 12) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACCCUGGGACGCAGAGCCGUGGCUGGCCUUCUGGCCUCCCCAAGCCCUGCCCAAGCCCAGACCUUGACUAGAGUGCCUAGACCGGCCGAACUCGCUCCCCUGUGCGGACGGAGGGGACUCAGGACUGACAUCGACGCAACAUGCACUCCAAGACGCGCCUCCAGCAACCAGCGGGGCCUCAACCAGAUUUGGAACGUGAAAAAGCAGUCCGUCUAUCUGAUGAACCUCCGCAAGUCCGGCACCUUGGGGCAUCCCGGGUCACUGGAUGAAACCACCUACGAACGGCUGGCCGAAGAGACUCUCGACUCCCUGGCCGAGUUCUUCGAGGACCUGGCGGAUAAGCCGUACACUUUCGAGGACUACGAUGUCUCUUUCGGAUCCGGCGUGCUGACCGUGAAGCUCGGUGGCGACCUCGGAACUUACGUGAUCAACAAGCAAACGCCCAACAAGCAGAUCUGGCUGUCCUCGCCGUCAUCGGGACCUAAGCGCUACGAUUGGACCGGGAAGAAUUGGGUGUACUCGCACGACGGUGUCAGCCUGCACGAGCUGCUUGCGGCGGAACUGACCAAGGCACUCAAGACCAAACUGGACCUGUCCAGCCUGGCCUACUCCGGAAAGGACGCCUAGGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU

In one embodiment, a codon-optimized human FXN mRNA sequence includesSEQ ID NO: 3.

In one embodiment, a full-length codon-optimized human FXN mRNA sequenceis:

(SEQ ID NO: 13) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACUCUGGGCCGGAGAGCUGUGGCAGGCCUUCUCGCCUCGCCAUCCCCUGCCCAAGCGCAGACCCUGACUAGGGUCCCUAGGCCUGCCGAGUUGGCACCGUUGUGCGGUCGGAGAGGACUGCGCACCGACAUCGAUGCCACCUGUACUCCUCGGAGAGCCUCGUCCAACCAGCGGGGCCUGAACCAGAUCUGGAACGUGAAGAAACAGUCCGUCUACCUGAUGAACCUCCGCAAGUCGGGAACCCUGGGACAUCCGGGUUCCCUGGAUGAGACUACGUACGAACGGCUGGCGGAAGAAACCCUGGACUCCCUGGCGGAGUUCUUCGAGGACCUGGCUGACAAGCCCUACACUUUUGAGGACUACGACGUGUCAUUCGGAAGCGGAGUGUUGACAGUGAAGCUGGGGGGCGAUCUGGGAACCUACGUGAUCAACAAGCAGACCCCGAACAAGCAAAUUUGGCUGUCCUCACCCUCCUCCGGACCUAAACGCUACGACUGGACCGGGAAGAACUGGGUGUAUAGCCACGACGGUGUCAGCCUUCACGAACUGCUUGCGGCCGAACUGACCAAGGCCCUCAAGACCAAGCUCGAUCUGUCUAGCCUCGCCUACUCCGGAAAGGACGCCUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

In one embodiment, another full-length codon-optimized human FXN mRNAsequence is:

(SEQ ID NO: 14) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACUCUGGGCCGGAGAGCUGUGGCAGGCCUUCUCGCCUCGCCAUCCCCUGCCCAAGCGCAGACCCUGACUAGGGUCCCUAGGCCUGCCGAGUUGGCACCGUUGUGCGGUCGGAGAGGACUGCGCACCGACAUCGAUGCCACCUGUACUCCUCGGAGAGCCUCGUCCAACCAGCGGGGCCUGAACCAGAUCUGGAACGUGAAGAAACAGUCCGUCUACCUGAUGAACCUCCGCAAGUCGGGAACCCUGGGACAUCCGGGUUCCCUGGAUGAGACUACGUACGAACGGCUGGCGGAAGAAACCCUGGACUCCCUGGCGGAGUUCUUCGAGGACCUGGCUGACAAGCCCUACACUUUUGAGGACUACGACGUGUCAUUCGGAAGCGGAGUGUUGACAGUGAAGCUGGGGGGCGAUCUGGGAACCUACGUGAUCAACAAGCAGACCCCGAACAAGCAAAUUUGGCUGUCCUCACCCUCCUCCGGACCUAAACGCUACGACUGGACCGGGAAGAACUGGGUGUAUAGCCACGACGGUGUCAGCCUUCACGAACUGCUUGCGGCCGAACUGACCAAGGCCCUCAAGACCAAGCUCGAUCUGUCUAGCCUCGCCUACUCCGGAAAGGACGCCUGAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU

In one embodiment, a codon-optimized human FXN mRNA sequence includesSEQ ID NO: 4.

In one embodiment, a full-length codon-optimized human FXN mRNA sequenceis:

(SEQ ID NO: 15) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACCUUGGGACGGAGAGCCGUGGCUGGACUGUUGGCCUCUCCUUCCCCGGCACAAGCCCAAACUCUGACCCGGGUCCCUAGACCGGCAGAGCUGGCUCCCCUGUGUGGUCGGCGGGGACUGAGAACUGAUAUUGACGCCACAUGCACUCCUAGGCGCGCGAGCUCCAAUCAGCGCGGCCUGAACCAGAUCUGGAACGUGAAGAAGCAGUCCGUCUACUUGAUGAACCUCCGCAAGUCCGGCACUCUGGGCCAUCCGGGAUCCCUCGACGAGACUACCUACGAGCGGCUGGCGGAAGAAACCCUGGAUUCCCUGGCCGAAUUCUUCGAGGACCUGGCCGACAAGCCCUACACCUUUGAGGACUACGACGUGUCCUUCGGAUCGGGAGUGCUGACCGUGAAGCUCGGCGGAGAUCUCGGGACUUAUGUGAUCAACAAGCAGACGCCGAACAAGCAGAUCUGGCUUAGCUCACCCUCGAGCGGACCAAAGCGCUACGACUGGACCGGCAAAAACUGGGUGUACUCCCACGAUGGUGUCAGCCUUCACGAACUGCUGGCCGCGGAACUGACCAAGGCCCUUAAGACCAAGCUCGACCUCUCAUCCCUGGCCUACUCCGGGAAAGACGCGUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

In one embodiment, another full-length codon-optimized human FXN mRNAsequence is:

(SEQ ID NO: 16) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGUGGACCUUGGGACGGAGAGCCGUGGCUGGACUGUUGGCCUCUCCUUCCCCGGCACAAGCCCAAACUCUGACCCGGGUCCCUAGACCGGCAGAGCUGGCUCCCCUGUGUGGUCGGCGGGGACUGAGAACUGAUAUUGACGCCACAUGCACUCCUAGGCGCGCGAGCUCCAAUCAGCGCGGCCUGAACCAGAUCUGGAACGUGAAGAAGCAGUCCGUCUACUUGAUGAACCUCCGCAAGUCCGGCACUCUGGGCCAUCCGGGAUCCCUCGACGAGACUACCUACGAGCGGCUGGCGGAAGAAACCCUGGAUUCCCUGGCCGAAUUCUUCGAGGACCUGGCCGACAAGCCCUACACCUUUGAGGACUACGACGUGUCCUUCGGAUCGGGAGUGCUGACCGUGAAGCUCGGCGGAGAUCUCGGGACUUAUGUGAUCAACAAGCAGACGCCGAACAAGCAGAUCUGGCUUAGCUCACCCUCGAGCGGACCAAAGCGCUACGACUGGACCGGCAAAAACUGGGUGUACUCCCACGAUGGUGUCAGCCUUCACGAACUGCUGGCCGCGGAACUGACCAAGGCCCUUAAGACCAAGCUCGACCUCUCAUCCCUGGCCUACUCCGGGAAAGACGCGUGAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU

SEQ ID NOs: 9-16 include 5′ and 3′ untranslated regions framing acodon-optimized hFXN-encoding mRNA.

In some embodiments, a suitable mRNA sequence encodes a homolog or ananalog of human frataxin. For example, a homolog or an analog of humanfrataxin may be a modified human frataxin protein containing one or moreamino acid substitutions, deletions, and/or insertions as compared to awild-type or naturally-occurring human frataxin while retainingsubstantial human frataxin activity. In some embodiments, an mRNAsuitable for the present invention encodes an amino acid sequence atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 5. In someembodiments, an mRNA suitable for the present invention encodes aprotein substantially identical to human frataxin. In some embodiments,an mRNA suitable for the present invention encodes an amino acidsequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 5. Insome embodiments, an mRNA suitable for the present invention encodes afragment or a portion of human frataxin protein. In some embodiments, anmRNA suitable for the present invention encodes a fragment or a portionof human frataxin protein, wherein the fragment or portion of theprotein still maintains frataxin activity similar to that of thewild-type protein. In some embodiments, an mRNA suitable for the presentinvention has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, an mRNAsuitable for the present invention comprises a nucleotide sequenceidentical to SEQ ID NO: 1. In some embodiments, an mRNA suitable for thepresent invention comprises a nucleotide sequence identical to SEQ IDNO: 2. In some embodiments, an mRNA suitable for the present inventioncomprises a nucleotide sequence identical to SEQ ID NO: 3. In someembodiments, an mRNA suitable for the present invention comprises anucleotide sequence identical to SEQ ID NO: 4.

In some embodiments, a suitable mRNA encodes a fusion protein comprisinga full length, fragment or portion of a human frataxin protein fused toanother protein (e.g., an N or C terminal fusion). In some embodiments,the protein fused to the mRNA encoding a full length, fragment orportion of a human frataxin protein encodes a signal or a cellulartargeting sequence.

Synthesis of mRNA

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence fordesired mRNA and a termination signal.

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., mRNAs encoding frataxin) includea 5′ cap structure. A 5′ cap is typically added as follows: first, anRNA terminal phosphatase removes one of the terminal phosphate groupsfrom the 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, mRNAs (e.g., mRNAs encoding frataxin) include a 3′poly(A) tail structure. A poly-A tail on the 3′ terminus of mRNAtypically includes about 10 to 300 adenosine nucleotides (e.g., about 10to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides,about 10 to 100 adenosine nucleotides, about 20 to 70 adenosinenucleotides, or about 20 to 60 adenosine nucleotides). In someembodiments, mRNAs include a 3′ poly(C) tail structure. A suitablepoly-C tail on the 3′ terminus of mRNA typically include about 10 to 200cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides,about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosinenucleotides). The poly-C tail may be added to the poly-A tail or maysubstitute the poly-A tail.

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Cap Structure

In some embodiments, mRNAs include a 5′ cap structure. A 5′ cap istypically added as follows: first, an RNA terminal phosphatase removesone of the terminal phosphate groups from the 5′ nucleotide, leaving twoterminal phosphates; guanosine triphosphate (GTP) is then added to theterminal phosphates via a guanylyl transferase, producing a 5′5′5triphosphate linkage; and the 7-nitrogen of guanine is then methylatedby a methyltransferase. Examples of cap structures include, but are notlimited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

Naturally occurring cap structures comprise a 7-methyl guanosine that islinked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in a dinucleotide cap of m⁷G(5′)ppp(5′)N, where Nis any nucleoside. In vivo, the cap is added enzymatically. The cap isadded in the nucleus and is catalyzed by the enzyme guanylyltransferase. The addition of the cap to the 5′ terminal end of RNAoccurs immediately after initiation of transcription. The terminalnucleoside is typically a guanosine, and is in the reverse orientationto all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription ism⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap intranscription with T7 RNA polymerase in vitro to obtain RNAs having acap structure in their 5′-termini. The prevailing method for the invitro synthesis of capped mRNA employs a pre-formed dinucleotide of theform m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator of transcription.

To date, a usual form of a synthetic dinucleotide cap used in in vitrotranslation experiments is the Anti-Reverse Cap Analog (“ARCA”) ormodified ARCA, which is generally a modified cap analog in which the 2′or 3′ OH group is replaced with —OCH3.

Additional cap analogs include, but are not limited to, a chemicalstructures selected from the group consisting of m⁷GpppG, m⁷GpppA,m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog(e.g., m^(2,7)GpppG), trimethylated cap analog (e.g., m^(2,2,7)GpppG),dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reversecap analogs (e.g., ARCA; m⁷,^(2′Ome)GpppG, m^(72′d)GpppG,m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives)(see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs withsuperior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”)linked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. Apreferred embodiment of a m⁷G cap utilized in embodiments of theinvention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a2′-O-methyl residue of the ribose attached to bases 1 and 2. In someembodiments, the cap is a Cap1 structure. Cap1 structures have a2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2structure. Cap2 structures have a 2′-O-methyl residue attached to bothbases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which arecommercially available. These include the m⁷GpppG described above, aswell as the ARCA 3′-OCH₃ and 2′-OCH₃ cap analogs (Jemielity, J. et al.,RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodimentsof the invention include N7-benzylated dinucleoside tetraphosphateanalogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),phosphorothioate cap analogs (described in Grudzien-Nogalska, E., etal., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylatedcap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529, eachof which are incorporated by reference herein for all purposes.

Tail Structure

Typically, the presence of a “tail” serves to protect the mRNA fromexonuclease degradation. The poly A tail is thought to stabilize naturalmessengers and synthetic sense RNA. Therefore, in certain embodiments along poly A tail can be added to an mRNA molecule thus rendering the RNAmore stable. Poly A tails can be added using a variety of art-recognizedtechniques. For example, long poly A tails can be added to synthetic orin vitro transcribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, mRNAs include a 3′ poly(A) tail structure.Typically, the length of the poly A tail can be at least about 10, 50,100, 200, 300, 400 at least 500 nucleotides. In some embodiments, apoly-A tail on the 3′ terminus of mRNA typically includes about 10 to300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides,about 10 to 150 adenosine nucleotides, about 10 to 100 adenosinenucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60adenosine nucleotides). In some embodiments, mRNAs include a 3′ poly(C)tail structure. A suitable poly-C tail on the 3′ terminus of mRNAtypically include about 10 to 200 cytosine nucleotides (e.g., about 10to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, orabout 10 to 40 cytosine nucleotides). The poly-C tail may be added tothe poly-A tail or may substitute the poly-A tail.

In some embodiments, the length of the poly A or poly C tail is adjustedto control the stability of a modified sense mRNA molecule of theinvention and, thus, the transcription of protein. For example, sincethe length of the poly A tail can influence the half-life of a sensemRNA molecule, the length of the poly A tail can be adjusted to modifythe level of resistance of the mRNA to nucleases and thereby control thetime course of polynucleotide expression and/or polypeptide productionin a target cell.

5′ and 3′ Untranslated Regions

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA moleculeswhich are stable (e.g., globin, actin, GAPDH, tubulin, histone, orcitric acid cycle enzymes) to increase the stability of the sense mRNAmolecule. For example, a 5′ UTR sequence may include a partial sequenceof a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improvethe nuclease resistance and/or improve the half-life of thepolynucleotide. Also contemplated is the inclusion of a sequenceencoding human growth hormone (hGH), or a fragment thereof to the 3′ endor untranslated region of the polynucleotide (e.g., mRNA) to furtherstabilize the polynucleotide. Generally, these modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of thepolynucleotide relative to their unmodified counterparts, and include,for example modifications made to improve such polynucleotides'resistance to in vivo nuclease digestion.

Modified mRNA

In some embodiments, mRNA according to the present invention may besynthesized as unmodified or modified mRNA. In some embodiments,unmodified mRNA comprises a 5′ cap, 5′ and 3′ UTRs and a polyA tail, butno modifications to the nucleotides of the mRNA. In some embodiments,modifications of mRNA can include modifications of the nucleotides ofthe RNA. A modified mRNA according to the invention can thus include,for example, backbone modifications, sugar modifications or basemodifications. In some embodiments, mRNAs may be synthesized fromnaturally occurring nucleotides and/or nucleotide analogues (modifiednucleotides) including, but not limited to, purines (adenine (A),guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), andas modified nucleotides analogues or derivatives of purines andpyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g., from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732,4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418,5,153,319, 5,262,530 and 5,700,642, the disclosures of each of which areincorporated by reference for all purposes.

In some embodiments, mRNAs (e.g., mRNAs encoding frataxin) may containRNA backbone modifications. Typically, a backbone modification is amodification in which the phosphates of the backbone of the nucleotidescontained in the RNA are modified chemically. Exemplary backbonemodifications typically include, but are not limited to, modificationsfrom the group consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g. cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., mRNAs encoding frataxin) may containsugar modifications. A typical sugar modification is a chemicalmodification of the sugar of the nucleotides it contains including, butnot limited to, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), azidotriphosphates(2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate) or 4′-thioribonucleotides (described in U.S. patentapplication US 2016/0031928, filed Sep. 14, 2015, incorporated herein byreference for all purposes).

In some embodiments, mRNAs (e.g., mRNAs encoding frataxin) may containmodifications of the bases of the nucleotides (base modifications). Amodified nucleotide which contains a base modification is also called abase-modified nucleotide. Examples of such base-modified nucleotidesinclude, but are not limited to, 2-amino-6-chloropurine riboside5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine5′-triphosphate, O6-methylguanosine 5′-triphosphate, pseudouridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

Delivery Vehicles

According to the present invention, mRNA encoding a frataxin protein(e.g., a full length, fragment, or portion of a frataxin protein) asdescribed herein may be delivered as naked RNA (unpackaged) or viadelivery vehicles. As used herein, the terms “delivery vehicle,”“transfer vehicle,” “nanoparticle” or grammatical equivalent, are usedinterchangeably.

Delivery vehicles can be formulated in combination with one or moreadditional nucleic acids, carriers, targeting ligands or stabilizingreagents, or in pharmacological compositions where it is mixed withsuitable excipients. Techniques for formulation and administration ofdrugs may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition. A particular deliveryvehicle is selected based upon its ability to facilitate thetransfection of a nucleic acid to a target cell.

In some embodiments, mRNAs encoding a frataxin protein may be deliveredvia a single delivery vehicle. In some embodiments, mRNAs encoding afrataxin protein may be delivered via one or more delivery vehicles eachof a different composition. According to various embodiments, suitabledelivery vehicles include, but are not limited to polymer basedcarriers, such as polyethyleneimine (PEI), lipid nanoparticles andliposomes, nanoliposomes, ceramide-containing nanoliposomes,proteoliposomes, both natural and synthetically-derived exosomes,natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates,calcium phosphor-silicate nanoparticulates, calcium phosphatenanoparticulates, silicon dioxide nanoparticulates, nanocrystallineparticulates, semiconductor nanoparticulates, poly(D-arginine),sol-gels, nanodendrimers, starch-based delivery systems, micelles,emulsions, niosomes, multi-domain-block polymers (vinyl polymers,polypropyl acrylic acid polymers, dynamic polyconjugates), dry powderformulations, plasmids, viruses, calcium phosphate nucleotides,aptamers, peptides and other vectorial tags. Also contemplated is theuse of bionanocapsules and other viral capsid proteins assemblies as asuitable transfer vehicle. (Hum. Gene Ther. 2008 September;19(9):887-95).

A delivery vehicle comprising FXN mRNA may be administered and dosed inaccordance with current medical practice, taking into account theclinical condition of the subject, the site and method of administration(e.g., local and systemic, including intrathecal and via injection), thescheduling of administration, the subject's age, sex, body weight, andother factors relevant to clinicians of ordinary skill in the art. The“effective amount” for the purposes herein may be determined by suchrelevant considerations as are known to those of ordinary skill inexperimental clinical research, pharmacological, clinical and medicalarts. In some embodiments, the amount administered is effective toachieve at least some stabilization, improvement or elimination ofsymptoms and other indicators as are selected as appropriate measures ofdisease progress, regression or improvement by those of skill in theart. For example, a suitable amount and dosing regimen is one thatcauses at least transient protein production.

In some embodiments, delivery vehicles are formulated such that they aresuitable for extended-release of the mRNA contained therein. Suchextended-release compositions may be conveniently administered to asubject at extended dosing intervals.

Liposomal Delivery Vehicles

In some embodiments, a suitable delivery vehicle is a liposomal deliveryvehicle, e.g., a liposome. As used herein, liposomal delivery vehicles,e.g., liposomes, are usually characterized as microscopic vesicleshaving an interior aqua space sequestered from an outer medium by amembrane of one or more bilayers. Bilayer membranes of liposomes aretypically formed by amphiphilic molecules, such as lipids of syntheticor natural origin that comprise spatially separated hydrophilic andhydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).Bilayer membranes of the liposomes can also be formed by amphophilicpolymers and surfactants (e.g., polymerosomes, niosomes, etc.). In thecontext of the present invention, a liposomal delivery vehicle typicallyserves to transport a desired mRNA to a target cell or tissue. In someembodiments, a nanoparticle delivery vehicle is a liposome. In someembodiments, a liposome comprises one or more cationic lipids, one ormore non-cationic lipids, one or more cholesterol-based lipids, one ormore PEG-modified lipids, and one or more sphingolipids. In someembodiments, a liposome comprises one or more cationic lipids, one ormore non-cationic lipids, one or more cholesterol-based lipids and oneor more PEG-modified lipids. In some embodiments, a liposome comprisesone or more cationic lipids, one or more non-cationic lipids, and one ormore PEG-modified lipids. In some embodiments, a liposome comprises nomore than three distinct lipid components. In some embodiments, onedistinct lipid component is a sterol-based cationic lipid.

Cationic Lipids

As used herein, the term “cationic lipids” refers to any of a number oflipid and lipidoid species that have a net positive charge at a selectedpH, such as at physiological pH. Several cationic lipids have beendescribed in the literature, many of which are commercially available.

Suitable cationic lipids for use in the compositions and methods of theinvention include the cationic lipids as described in InternationalPatent Publication WO 2010/144740, which is incorporated herein byreference for all purposes. In certain embodiments, the compositions andmethods of the present invention include a cationic lipid,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include ionizable cationic lipids as describedin International Patent Publication WO 2013/149140, which isincorporated herein by reference for all purposes. In some embodiments,the compositions and methods of the present invention include a cationiclipid of one of the following formulas:

or a pharmaceutically acceptable salt thereof, wherein R₁ and R₂ areeach independently selected from the group consisting of hydrogen, anoptionally substituted, variably saturated or unsaturated C₁-C₂₀ alkyland an optionally substituted, variably saturated or unsaturated C₆-C₂₀acyl; wherein L₁ and L₂ are each independently selected from the groupconsisting of hydrogen, an optionally substituted C₁-C₃₀ alkyl, anoptionally substituted variably unsaturated C₁-C₃₀ alkenyl, and anoptionally substituted C₁-C₃₀ alkynyl; wherein m and o are eachindependently selected from the group consisting of zero and anypositive integer (e.g., where m is three); and wherein n is zero or anypositive integer (e.g., where n is one). In certain embodiments, thecompositions and methods of the present invention include the cationiclipid (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (“HGT5000”), having a compound structureof:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include thecationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (“HGT5001”, also called “CCBene”, whichcan be used interchangeably), having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include thecationic lipid and(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (“HGT5002”), having a compound structureof:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include cationic lipids described as aminoalcohollipidoids in International Patent Publication WO 2010/053572, which isincorporated herein by reference for all purposes. In certainembodiments, the compositions and methods of the present inventioninclude a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2016/118725, which is incorporatedherein by reference for all purposes. In certain embodiments, thecompositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2016/118724, which is incorporatedherein by reference for all purposes. In certain embodiments, thecompositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include a cationic lipid having the formula of14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, andpharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publications WO 2013/063468 and WO 2016/205691,each of which are incorporated herein by reference for all purposes. Insome embodiments, the compositions and methods of the present inventioninclude a cationic lipid of the following formula:

or pharmaceutically acceptable salts thereof, wherein each instance ofR^(L) is independently optionally substituted C₆-C₄₀ alkenyl. In certainembodiments, the compositions and methods of the present inventioninclude a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2015/184256, which is incorporatedherein by reference for all purposes. In some embodiments, thecompositions and methods of the present invention include a cationiclipid of the following formula:

or a pharmaceutically acceptable salt thereof, wherein each Xindependently is O or S; each Y independently is O or S; each mindependently is 0 to 20; each n independently is 1 to 6; each R_(A) isindependently hydrogen, optionally substituted C1-50 alkyl, optionallysubstituted C2-50 alkenyl, optionally substituted C2-50 alkynyl,optionally substituted C3-10 carbocyclyl, optionally substituted 3-14membered heterocyclyl, optionally substituted C6-14 aryl, optionallysubstituted 5-14 membered heteroaryl or halogen; and each R_(B) isindependently hydrogen, optionally substituted C1-50 alkyl, optionallysubstituted C2-50 alkenyl, optionally substituted C2-50 alkynyl,optionally substituted C3-10 carbocyclyl, optionally substituted 3-14membered heterocyclyl, optionally substituted C6-14 aryl, optionallysubstituted 5-14 membered heteroaryl or halogen. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “Target 23”, having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2016/004202, which is incorporatedherein by reference for all purposes. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

or a pharmaceutically acceptable salt thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include the cationic lipids as described in J.McClellan, M. C. King, Cell 2010, 141, 210-217 and in Whitehead et al.,Nature Communications (2014) 5:4277, which is incorporated herein byreference for all purposes. In certain embodiments, the cationic lipidsof the compositions and methods of the present invention include acationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2015/199952, which is incorporatedherein by reference for all purposes. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/004143, which is incorporatedherein by reference for all purposes. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/075531, which is incorporatedherein by reference for all purposes. In some embodiments, thecompositions and methods of the present invention include a cationiclipid of the following formula:

or a pharmaceutically acceptable salt thereof, wherein one of L¹ or L²is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x), —S—S—, —C(═O)S—,—SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)—, or —NR^(a)C(═O)O—; and the other of L¹ or L² is—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x), —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond; G¹ and G² are each independentlyunsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G³ is C₁-C₂₄alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene;R^(a) is H or C₁-C₁₂ alkyl; R¹ and R² are each independently C₆-C₂₄alkyl or C₆-C₂₄ alkenyl; R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or—NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H or C₁-C₆ alkyl; and x is 0, 1or 2.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/117528, which is incorporatedherein by reference for all purposes. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, thecompositions and methods of the present invention include a cationiclipid having the compound structure:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/049245, which is incorporatedherein by reference for all purposes. In some embodiments, the cationiclipids of the compositions and methods of the present invention includea compound of one of the following formulas:

and pharmaceutically acceptable salts thereof. For any one of these fourformulas, R₄ is independently selected from —(CH₂)_(n)Q and—(CH₂)_(n)CHQ(R); Q is selected from the group consisting of —OR, —OH,—O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle; and n is 1, 2, or 3. In certain embodiments, thecompositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include the cationic lipids as described inInternational Patent Publication WO 2017/173054 and WO 2015/095340, eachof which is incorporated herein by reference for all purposes. Incertain embodiments, the compositions and methods of the presentinvention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the invention include cholesterol-based cationic lipids. In certainembodiments, the compositions and methods of the present inventioninclude imidazole cholesterol ester or “ICE”, having a compoundstructure of:

and pharmaceutically acceptable salts thereof.

Other suitable cationic lipids for use in the compositions and methodsof the present invention include cleavable cationic lipids as describedin International Patent Publication WO 2012/170889, which isincorporated herein by reference for all purposes. In some embodiments,the compositions and methods of the present invention include a cationiclipid of the following formula:

wherein R₁ is selected from the group consisting of imidazole,guanidinium, amino, imine, enamine, an optionally-substituted alkylamino (e.g., an alkyl amino such as dimethylamino) and pyridyl; whereinR₂ is selected from the group consisting of one of the following twoformulas:

and wherein R₃ and R₄ are each independently selected from the groupconsisting of an optionally substituted, variably saturated orunsaturated C₆-C₂₀ alkyl and an optionally substituted, variablysaturated or unsaturated C₆-C₂₀ acyl; and wherein n is zero or anypositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty or more). In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4001”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4002”, having a compound structure of:

-   -   (HGT4002)        and pharmaceutically acceptable salts thereof. In certain        embodiments, the compositions and methods of the present        invention include a cationic lipid, “HGT4003”, also called        “DLin-SS-DMA” which can be used interchangeably, having a        compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid, “HGT4004”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In certain embodiments,the compositions and methods of the present invention include a cationiclipid “HGT4005”, having a compound structure of:

and pharmaceutically acceptable salts thereof.

In some embodiments, the compositions and methods of the presentinvention include the cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”).(Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No.4,897,355, each of which are incorporated herein by reference for allpurposes). Other cationic lipids suitable for the compositions andmethods of the present invention include, for example,5-carboxyspermylglycinedioctadecylamide (“DOGS”);2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium(“DOSPA”) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), U.S.Pat. Nos. 5,171,678; 5,334,761; each of which are incorporated herein byreference for all purposes); 1,2-Dioleoyl-3-Dimethylammonium-Propane(“DODAP”); 1,2-Dioleoyl-3-Trimethylammonium-Propane (“DOTAP”).

Additional exemplary cationic lipids suitable for the compositions andmethods of the present invention also include:1,2-distearyloxy-N,N-dimethyl-3-aminopropane (“DSDMA”);1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (“DODMA”);1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLinDMA”);1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (“DLenDMA”);N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”);3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(“CLinDMA”); 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane (“CpLinDMA”);N,N-dimethyl-3,4-dioleyloxybenzylamine (“DMOBA”);1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (“DOcarbDAP”);2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (“DLinDAP”);1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (“DLincarbDAP”);1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (“DLinCDAP”);2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (“DLin-K-DMA”);2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9, 12-dien-1-yloxy]propane-1-amine (“Octyl-CLinDMA”);(2R)-2((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA (2R)”);(2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyh3-[(9Z,12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine (“Octyl-CLinDMA (2S)”);2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (“DLin-K-XTC2-DMA”);and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(“DLin-KC2-DMA”) (see, WO 2010/042877, which is incorporated herein byreference for all purposes; Semple et al., Nature Biotech. 28: 172-176(2010), which is incorporated herein by reference for all purposes).(Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey,D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); InternationalPatent Publication WO 2005/121348; each of which are incorporated hereinby reference for all purposes). In some embodiments, one or more of thecationic lipids comprise at least one of an imidazole, dialkylamino, orguanidinium moiety.

In some embodiments, one or more cationic lipids suitable for thecompositions and methods of the present invention include2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (“XTC”);(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (“ALNY-100”) and/or4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide(“NC98-5”).

In some embodiments, the compositions of the present invention includeone or more cationic lipids that constitute at least about 5%, 10%, 20%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, ofthe total lipid content in the composition, e.g., a lipid nanoparticle.In some embodiments, the compositions of the present invention includeone or more cationic lipids that constitute at least about 5%, 10%, 20%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, ofthe total lipid content in the composition, e.g., a lipid nanoparticle.In some embodiments, the compositions of the present invention includeone or more cationic lipids that constitute about 30-70% (e.g., about30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about30-40%, about 35-50%, about 35-45%, or about 35-40%), measured byweight, of the total lipid content in the composition, e.g., a lipidnanoparticle. In some embodiments, the compositions of the presentinvention include one or more cationic lipids that constitute about30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%,about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%), measured as mol %, of the total lipid content in thecomposition, e.g., a lipid nanoparticle.

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic(“helper”) lipids. As used herein, the phrase “non-cationic lipid”refers to any neutral, zwitterionic or anionic lipid. As used herein,the phrase “anionic lipid” refers to any of a number of lipid speciesthat carry a net negative charge at a selected H, such as physiologicalpH. In some embodiments, a non-cationic lipid is a neutral lipid, i.e.,a lipid that does not carry a net charge in the conditions under whichthe composition is formulated and/or administered. Non-cationic lipidsinclude, but are not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine(DOPE), palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine,sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, such non-cationic lipids may be used alone, but arepreferably used in combination with other lipids, for example, cationiclipids. In some embodiments, the non-cationic lipid may comprise a molarratio of about 5% to about 90%, about 10% to about 70%, about 15% toabout 40%, or about 20% to about 35% of the total lipid present in aliposome. In some embodiments, the percentage of non-cationic lipid in aliposome may be greater than 5%, greater than 10%, greater than 20%,greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or morecholesterol-based lipids. Suitable cholesterol-based cationic lipidsinclude, for example, DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335; each of which are incorporated herein byreference for all purposes), or ICE. In some embodiments, thecholesterol-based lipid may comprise a molar ratio of about 2% to about70%, about 5% to about 65%, about 10% to about 60%, about 15% to about50%, or about 20% to about 40% of the total lipid present in a liposome.In some embodiments, the percentage of cholesterol-based lipid in theliposome may be greater than 5%, greater than 10%, greater than 20%,greater than 30%, greater than 40%, greater than 50%, or greater than60%.

PEG-Modified Lipids

The use of polyethylene glycol (PEG)-modified phospholipids andderivatized lipids such as derivatized cerarmides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention,either alone or preferably in combination with other lipid formulationstogether which comprise the transfer vehicle (e.g., a liposome).Contemplated PEG-modified lipids include, but are not limited to, apolyethylene glycol chain of up to S kDa in length covalently attachedto a lipid with alkyl chain(s) of C₆-C₂₀ length. The addition of suchcomponents may prevent complex aggregation and may also provide a meansfor increasing circulation lifetime and increasing the delivery of thelipid-nucleic acid composition to the target tissues, (Klibanov et al.(1990) FEBS Letters, 268 (1): 235-237), or they may be selected torapidly exchange out of the formulation in vivo (see U.S. Pat. No.5,885,613). Particularly useful exchangeable lipids are PEG-ceramideshaving shorter acyl chains (e.g., C₁₄ or C₁₈). A PEG-modifiedphospholipid and derivitized lipids of the present invention maycomprise a molar ratio from about 0% to about 20%, about 0.5% to about20%, about 1% to about 15%, about 4% to about 10%, or about 2% of thetotal lipid present in the liposomal transfer vehicle.

Sphingolipids

In some embodiments, provided liposomes comprise one or moresphingolipids. Suitable sphingolipids include, for example, sphingosine,ceramide, sphingomyelin, cerebroside and ganglioside. In someembodiments, sphingomyelin is a brain sphingomyelin. In someembodiments, the one or more sphingolipids may comprise a molar ratio ofabout 5% to about 30%, about 10% to about 25%, or about 15% to about 20%of the total lipid present in a liposome. In some embodiments, thepercentage of sphingolipid in the liposome may be greater than 1%,greater than 5%, greater than 10%, greater than 15%, or greater than20%.

According to various embodiments, the selection of cationic lipids,non-cationic lipids, PEG-modified lipids and/or sphingolipids whichcomprise the liposome, as well as the relative molar ratio of suchlipids to each other, is based upon the characteristics of the selectedlipid(s), the nature of the intended target cells, the characteristicsof the mRNA to be delivered. Additional considerations include, forexample, the saturation of the alkyl chain, as well as the size, charge,pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus themolar ratios may be adjusted accordingly.

Polymers

In some embodiments, a suitable delivery vehicle is formulated using apolymer as a carrier, alone or in combination with other carriersincluding various lipids described herein. Thus, in some embodiments,liposomal delivery vehicles, as used herein, also encompassnanoparticles comprising polymers. Suitable polymers may include, forexample, polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PLL, PEGylated PLL and polyethyleneimine(PEI). When PEI is present, it may be branched PEI of a molecular weightranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

In some embodiments, the compositions and methods of the presentinvention include a cationic polymer (“PCMMA”) of the following formula:

-   -   wherein        -   R¹ is C₆-C₂₀ alkyl, C₆-C₂₀ alkenyl, or C₆-C₂₀ alkynyl;        -   L¹ is C₂-C₂₀ alkylene;        -   B¹ is NR²R³ or a 5- to 10-membered heteroaryl group;        -   R² is hydrogen or C₁-C₂₀ alkyl;        -   R³ is hydrogen, C₁-C₂₀ alkyl, or an N-protecting group;        -   or R² and R³, together with the nitrogen to which they are            attached, combine to form a 5- to 10-membered heterocyclic            group;        -   x is an integer of 5 to 500; and        -   y is an integer of 5 to 500.            and pharmaceutically acceptable salts thereof.

Additional Combinations

A suitable liposome for the present invention may include one or more ofany of the cationic lipids, non-cationic lipids, cholesterol lipids,PEG-modifed lipids, sphingolipids and/or polymers described herein atvarious ratios. As non-limiting examples, a suitable liposomeformulation may include a combination selected from cKK-E12, DOPE,cholesterol and DMG-PEG2K; C12-200, DOPE, cholesterol and DMG-PEG2K;HGT4003, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol andDMG-PEG2K; ICE, DOPE, and DMG-PEG2K; or cKK-E12, DOPE, cholesterol andDMG-PEG2K, and brain sphingomyelin.

In various embodiments, cationic lipids (e.g., cKK-E12, C12-200, ICE,and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about35-40%) of the liposome by molar ratio. In some embodiments, thepercentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/orHGT4003) is or greater than about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, or about 60% of the liposome by molar ratio.

In some embodiments, the ratio of cationic lipid(s) to non-cationiclipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may bebetween about 30-60:25-35:20-30:1-15, respectively. In some embodiments,the ratio of cationic lipid(s) to non-cationic lipid(s) tocholesterol-based lipid(s) to PEG-modified lipid(s) is approximately40:30:20:10, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEG-modified lipid(s) is approximately 40:30:25:5, respectively. In someembodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) tocholesterol-based lipid(s) to PEG-modified lipid(s) is approximately40:32:25:3, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEG-modified lipid(s) is approximately 50:25:20:5. In some embodiments,the ratio of sterol lipid(s) to non-cationic lipid(s) to PEG-modifiedlipid(s) is 50:45:5. In some embodiments, the ratio of sterol lipid(s)to non-cationic lipid(s) to PEG-modified lipid(s) is 50:40:10. In someembodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) toPEG-modified lipid(s) is 55:40:5. In some embodiments, the ratio ofsterol lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is55:35:10. In some embodiments, the ratio of sterol lipid(s) tonon-cationic lipid(s) to PEG-modified lipid(s) is 60:35:5. In someembodiments, the ratio of sterol lipid(s) to non-cationic lipid(s) toPEG-modified lipid(s) is 60:30:10. In some embodiments, the ratio ofcationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s)to PEG-modified lipid(s) to sphingolipid(s) may be between about30-50:10-30:10-30:1-15:5-25, respectively. In some embodiments, theratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-basedlipid(s) to PEG-modified lipid(s) to sphingolipid(s) is approximately35:25:20:10:10, respectively. In some embodiments, the ratio of cationiclipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) toPEG-modified lipid(s) to sphingolipid(s) is approximately 40:20:20:5:15,respectively. In some embodiments, the ratio of cationic lipid(s) tonon-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modifiedlipid(s) to sphingolipid(s) is approximately 45:15:25:10:5,respectively.

In some embodiments, a suitable liposome for the present inventioncomprises ICE and DOPE at an ICE:DOPE molar ratio of >1:1. In someembodiments, the ICE:DOPE molar ratio is <2.5:1. In some embodiments,the ICE:DOPE molar ratio is between 1:1 and 2.5:1. In some embodiments,the ICE:DOPE molar ratio is approximately 1.5:1. In some embodiments,the ICE:DOPE molar ratio is approximately 1.7:1. In some embodiments,the ICE:DOPE molar ratio is approximately 2:1. In some embodiments, asuitable liposome for the present invention comprises ICE and DMG-PEG-2Kat an ICE:DMG-PEG-2K molar ratio of >10:1. In some embodiments, theICE:DMG-PEG-2K molar ratio is <16:1. In some embodiments, theICE:DMG-PEG-2K molar ratio is approximately 12:1. In some embodiments,the ICE:DMG-PEG-2K molar ratio is approximately 14:1. In someembodiments, a suitable liposome for the present invention comprisesDOPE and DMG-PEG-2K at a DOPE: DMG-PEG-2K molar ratio of >5:1. In someembodiments, the DOPE: DMG-PEG-2K molar ratio is <11:1. In someembodiments, the DOPE: DMG-PEG-2K molar ratio is approximately 7:1. Insome embodiments, the DOPE: DMG-PEG-2K molar ratio is approximately10:1. In some embodiments, a suitable liposome for the present inventioncomprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratioof 50:45:5. In some embodiments, a suitable liposome for the presentinvention comprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2Kmolar ratio of 50:40:10. In some embodiments, a suitable liposome forthe present invention comprises ICE, DOPE and DMG-PEG-2K at anICE:DOPE:DMG-PEG-2K molar ratio of 55:40:5. In some embodiments, asuitable liposome for the present invention comprises ICE, DOPE andDMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratio of 55:35:10. In someembodiments, a suitable liposome for the present invention comprisesICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratio of60:35:5. In some embodiments, a suitable liposome for the presentinvention comprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2Kmolar ratio of 60:30:10.

Ratio of Distinct Lipid Components

In embodiments where a liposome comprises three and no more than threedistinct components of lipids, the ratio of total lipid content (i.e.,the ratio of lipid component (1):lipid component (2):lipid component(3)) can be represented as x:y:z, wherein(y+z)=100−x.

In some embodiments, each of “x,” “y,” and “z” represents molarpercentages of the three distinct components of lipids, and the ratio isa molar ratio.

In some embodiments, each of “x,” “y,” and “z” represents weightpercentages of the three distinct components of lipids, and the ratio isa weight ratio.

In some embodiments, lipid component (1), represented by variable “x,”is a sterol-based cationic lipid.

In some embodiments, lipid component (2), represented by variable “y,”is a helper lipid.

In some embodiments, lipid component (3), represented by variable “z” isa PEG lipid.

In some embodiments, variable “x,” representing the molar percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95%.

In some embodiments, variable “x,” representing the molar percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is no morethan about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about20%, or about 10%. In embodiments, variable “x” is no more than about65%, about 60%, about 55%, about 50%, about 40%.

In some embodiments, variable “x,” representing the molar percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is: at leastabout 50% but less than about 95%; at least about 50% but less thanabout 90%; at least about 50% but less than about 85%; at least about50% but less than about 80%; at least about 50% but less than about 75%;at least about 50% but less than about 70%; at least about 50% but lessthan about 65%; or at least about 50% but less than about 60%. Inembodiments, variable “x” is at least about 50% but less than about 70%;at least about 50% but less than about 65%; or at least about 50% butless than about 60%.

In some embodiments, variable “x,” representing the weight percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95%.

In some embodiments, variable “x,” representing the weight percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is no morethan about 95%, about 90%, about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about20%, or about 10%. In embodiments, variable “x” is no more than about65%, about 60%, about 55%, about 50%, about 40%.

In some embodiments, variable “x,” representing the weight percentage oflipid component (1) (e.g., a sterol-based cationic lipid), is: at leastabout 50% but less than about 95%; at least about 50% but less thanabout 90%; at least about 50% but less than about 85%; at least about50% but less than about 80%; at least about 50% but less than about 75%;at least about 50% but less than about 70%; at least about 50% but lessthan about 65%; or at least about 50% but less than about 60%. Inembodiments, variable “x” is at least about 50% but less than about 70%;at least about 50% but less than about 65%; or at least about 50% butless than about 60%.

In some embodiments, variable “z,” representing the molar percentage oflipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments,variable “z,” representing the molar percentage of lipid component (3)(e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. Inembodiments, variable “z,” representing the molar percentage of lipidcomponent (3) (e.g., a PEG lipid) is about 1% to about 10%, about 2% toabout 10%, about 3% to about 10%, about 4% to about 10%, about 1% toabout 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.

In some embodiments, variable “z,” representing the weight percentage oflipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments,variable “z,” representing the weight percentage of lipid component (3)(e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. Inembodiments, variable “z,” representing the weight percentage of lipidcomponent (3) (e.g., a PEG lipid) is about 1% to about 10%, about 2% toabout 10%, about 3% to about 10%, about 4% to about 10%, about 1% toabout 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.

For compositions having three and only three distinct lipid components,variables “x,” “y,” and “z” may be in any combination so long as thetotal of the three variables sums to 100% of the total lipid content.

Formation of Liposomes Encapsulating mRNA

The liposomal transfer vehicles for use in the compositions of theinvention can be prepared by various techniques which are presentlyknown in the art. The liposomes for use in provided compositions can beprepared by various techniques which are presently known in the art. Forexample, multilamellar vesicles (MLV) may be prepared according toconventional techniques, such as by depositing a selected lipid on theinside wall of a suitable container or vessel by dissolving the lipid inan appropriate solvent, and then evaporating the solvent to leave a thinfilm on the inside of the vessel or by spray drying. An aqueous phasemay then be added to the vessel with a vortexing motion which results inthe formation of MLVs. Unilamellar vesicles (ULV) can then be formed byhomogenization, sonication or extrusion of the multilamellar vesicles.In addition, unilamellar vesicles can be formed by detergent removaltechniques.

In certain embodiments, provided compositions comprise a liposomewherein the mRNA is associated on both the surface of the liposome andencapsulated within the same liposome. For example, during preparationof the compositions of the present invention, cationic liposomes mayassociate with the mRNA through electrostatic interactions. For example,during preparation of the compositions of the present invention,cationic liposomes may associate with the mRNA through electrostaticinteractions.

In some embodiments, the methods and compositions of the inventioncomprise mRNA encapsulated in a liposome. In some embodiments, the oneor more mRNA species may be encapsulated in the same liposome. In someembodiments, the one or more mRNA species may be encapsulated indifferent liposomes. In some embodiments, the mRNA is encapsulated inone or more liposomes, which differ in their lipid composition, molarratio of lipid components, size, charge (zeta potential), targetingligands and/or combinations thereof. In some embodiments, the one ormore liposome may have a different composition of sterol-based cationiclipids, neutral lipid, PEG-modified lipid and/or combinations thereof.In some embodiments the one or more liposomes may have a different molarratio of cholesterol-based cationic lipid, neutral lipid, andPEG-modified lipid used to create the liposome. In some embodiments, theone or more liposomes may have a different molar ratio of cationiclipid, neutral lipid, cholesterol-based lipid, PEG-modified lipid andsphingolipid used to create the liposome.

The process of incorporation of a desired mRNA into a liposome is oftenreferred to as “loading”. Exemplary methods are described in Lasic, etal., FEBS Lett., 312: 255-258, 1992, which is incorporated herein byreference for all purposes. The liposome-incorporated nucleic acids maybe completely or partially located in the interior space of theliposome, within the bilayer membrane of the liposome, or associatedwith the exterior surface of the liposome membrane. The incorporation ofa nucleic acid into liposomes is also referred to herein as“encapsulation” wherein the nucleic acid is entirely contained withinthe interior space of the liposome. The purpose of incorporating an mRNAinto a transfer vehicle, such as a liposome, is often to protect thenucleic acid from an environment which may contain enzymes or chemicalsthat degrade nucleic acids and/or systems or receptors that cause therapid excretion of the nucleic acids. Accordingly, in some embodiments,a suitable delivery vehicle is capable of enhancing the stability of themRNA contained therein and/or facilitate the delivery of mRNA to thetarget cell or tissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be madein various sizes. In some embodiments, provided liposomes may be madesmaller than previously known mRNA encapsulating liposomes. In someembodiments, decreased size of liposomes is associated with moreefficient delivery of mRNA. Selection of an appropriate liposome sizemay take into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made.

In some embodiments, an appropriate size of lipo some is selected tofacilitate systemic distribution of the protein encoded by the mRNA. Insome embodiments, it may be desirable to limit transfection of the mRNAto certain cells or tissues. For example, to target hepatocytes aliposome may be sized such that its dimensions are smaller than thefenestrations of the endothelial layer lining hepatic sinusoids in theliver; in such cases the liposome could readily penetrate suchendothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that thedimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,a liposome may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the liposome particle. In some embodiments, asuitable liposome has a size no greater than about 250 nm (e.g., nogreater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75nm, or 50 nm). In some embodiments, a suitable liposome has a sizeranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm).In some embodiments, a suitable liposome has a size ranging from about100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm,100-150 nm). In some embodiments, a suitable liposome has a size rangingfrom about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70nm, 10-60 nm, or 10-50 nm). In a particular embodiment, a suitableliposome has a size less than about 100 nm.

A variety of alternative methods known in the art are available forsizing of a population of liposomes. One such sizing method is describedin U.S. Pat. No. 4,737,323, incorporated herein by reference for allpurposes. Sonicating a liposome suspension either by bath or probesonication produces a progressive size reduction down to small ULV lessthan about 0.05 microns in diameter. Homogenization is another methodthat relies on shearing energy to fragment large liposomes into smallerones. In a typical homogenization procedure, MLV are recirculatedthrough a standard emulsion homogenizer until selected liposome sizes,typically between about 0.1 and 0.5 microns, are observed. The size ofthe liposomes may be determined by quasi-electric light scattering(QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng.,10:421-150 (1981), incorporated herein by reference for all purposes.Average liposome diameter may be reduced by sonication of formedliposomes. Intermittent sonication cycles may be alternated with QELSassessment to guide efficient liposome synthesis.

Pharmaceutical Compositions

To facilitate expression of mRNA in vivo, delivery vehicles such asliposomes can be formulated in combination with one or more additionalnucleic acids, carriers, targeting ligands or stabilizing reagents, orin pharmacological compositions where it is mixed with suitableexcipients. Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

Administration and Delivery

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, or intranasal. In particular embodiments,the intramuscular administration is to a muscle selected from the groupconsisting of skeletal muscle, smooth muscle and cardiac muscle. In someembodiments the administration results in delivery of the mRNA to amuscle cell. In some embodiments the administration results in deliveryof the mRNA to a hepatocyte (i.e., liver cell). In a particularembodiment, the intramuscular administration results in delivery of themRNA to a muscle cell.

Alternatively or additionally, liposomally encapsulated mRNAs andcompositions of the invention may be administered in a local rather thansystemic manner, for example, via injection of the pharmaceuticalcomposition directly into a targeted tissue, preferably in a sustainedrelease formulation. Local delivery can be affected in various ways,depending on the tissue to be targeted. For example, aerosols containingcompositions of the present invention can be inhaled (for nasal,tracheal, or bronchial delivery); compositions of the present inventioncan be injected into the site of injury, disease manifestation, or pain,for example; compositions can be provided in lozenges for oral,tracheal, or esophageal application; can be supplied in liquid, tabletor capsule form for administration to the stomach or intestines, can besupplied in suppository form for rectal or vaginal application; or caneven be delivered to the eye by use of creams, drops, or even injection.Formulations containing provided compositions complexed with therapeuticmolecules or ligands can even be surgically administered, for example inassociation with a polymer or other structure or substance that canallow the compositions to diffuse from the site of implantation tosurrounding cells. Alternatively, they can be applied surgically withoutthe use of polymers or supports.

mRNAs or mRNA-containing delivery vehicles (e.g., liposomes) asdescribed herein, are suitable for CNS delivery. In some embodiments,mRNA-loaded liposomes can be delivered to the CNS via various techniquesand routes including, but not limited to, intraparenchymal,intracerebral, intracerebroventricular (ICV), intrathecal (IT e.g.,IT-Lumbar, IT-cistema magna, etc,) administrations and any othertechniques and routes for injection directly or indirectly to the CNSand/or CSF.

In some embodiments, mRNA containing delivery vehicles (e.g., liposomes)are delivered to the CNS by injecting into the cerebrospinal fluid (CSF)of a subject in need of treatment. In some embodiments, intrathecaldelivery (also referred to as “intrathecal administration” or“intrathecal injection”) is used for delivering mRNA or mRNA-loadedliposomes into the CSF. As used herein, intrathecal delivery refers toan injection into the spinal canal (intrathecal space surrounding thespinal cord). Various techniques may be used including, withoutlimitation, lateral cerebroventricular injection through a burrhole orcistemal or lumbar puncture or the like. Exemplary methods are describedin Lazorthes et al., Advances in Drug Delivery Systems and Applicationsin Neurosurgery, 143-192 and in Omaya et al., Cancer Drug Delivery, 1:169-179, the contents of each of which are incorporated herein byreference for all purposes.

According to the present invention, mRNA or mRNA loaded liposomes may beinjected at any region surrounding the spinal canal. In someembodiments, mRNA or mRNA loaded liposomes are injected into the lumbararea or the cisterna magna or intraventricularly into a cerebralventricle space. As used herein, the term “lumbar region” or “lumbararea” refers to the area between the third and fourth lumbar (lowerback) vertebrae and, more inclusively, the L2-S1 region of the spine.Typically, intrathecal injection via the lumbar region or lumber area isalso referred to as “lumbar IT delivery” or “lumbar IT administration.”The term “cisterna magna” refers to the space around and below thecerebellum via the opening between the skull and the top of the spine.Typically, intrathecal injection via cisterna magna is also referred toas “cisterna magna delivery.” The term “cerebral ventricle” refers tothe cavities in the brain that are continuous with the central canal ofthe spinal cord. Typically, injections via the cerebral ventriclecavities are referred to as intracerebroventricular (ICV) delivery.

In some embodiments, “intrathecal administration” or “intrathecaldelivery” according to the present invention refers to lumbar ITadministration or delivery, for example, delivered between the third andfourth lumbar (lower back) vertebrae and, more inclusively, the L2-S1region of the spine.

In some embodiments, intrathecal delivery may be performed by eitherlumbar puncture (i.e., slow bolus) or via a port-catheter deliverysystem (i.e., infusion or bolus). In some embodiments, the catheter isinserted between the laminae of the lumbar vertebrae and the tip isthreaded up the thecal space to the desired level (generally L3-L4).

Provided methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of thetherapeutic agents (e.g., mRNA encoding frataxin) described herein.Therapeutic agents can be administered at regular intervals, dependingon the nature, severity and extent of the subject's condition (e.g.,Friedreich's ataxia). In some embodiments, a therapeutically effectiveamount of the therapeutic agents (e.g., mRNA encoding a frataxin) of thepresent invention may be administered intrathecally periodically atregular intervals (e.g., once every year, once every six months, onceevery five months, once every three months, bimonthly (once every twomonths), monthly (once every month), biweekly (once every two weeks),twice a month, once every 30 days, once every 28 days, once every 14days, once every 10 days, once every 7 days, weekly, twice a week,daily, or continuously).

In some embodiments, the CNS disease is associated with peripheralsymptoms. Thus, in some embodiments, intrathecal administration may beused in conjunction with other routes of administration (e.g.,intravenous, subcutaneously, intramuscularly, parenterally,transdermally, or transmucosally (e.g., orally or nasally)).

As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of mRNA contained in thepharmaceutical compositions of the present invention. Generally, atherapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating Friedreich's ataxia). For example, a therapeuticallyeffective amount may be an amount sufficient to achieve a desiredtherapeutic and/or prophylactic effect. Generally, the amount of mRNAadministered to a subject in need thereof will depend upon thecharacteristics of the subject. Such characteristics include thecondition, disease severity, general health, age, sex and body weight ofthe subject. One of ordinary skill in the art will be readily able todetermine appropriate dosages depending on these and other relatedfactors. In addition, both objective and subjective assays mayoptionally be employed to identify optimal dosage ranges.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg body weight to 500 mg/kg body weight, e.g., from about0.005 mg/kg body weight to 400 mg/kg body weight, from about 0.005 mg/kgbody weight to 300 mg/kg body weight, from about 0.005 mg/kg body weightto 200 mg/kg body weight, from about 0.005 mg/kg body weight to 100mg/kg body weight, from about 0.005 mg/kg body weight to 90 mg/kg bodyweight, from about 0.005 mg/kg body weight to 80 mg/kg body weight, fromabout 0.005 mg/kg body weight to 70 mg/kg body weight, from about 0.005mg/kg body weight to 60 mg/kg body weight, from about 0.005 mg/kg bodyweight to 50 mg/kg body weight, from about 0.005 mg/kg body weight to 40mg/kg body weight, from about 0.005 mg/kg body weight to 30 mg/kg bodyweight, from about 0.005 mg/kg body weight to 25 mg/kg body weight, fromabout 0.005 mg/kg body weight to 20 mg/kg body weight, from about 0.005mg/kg body weight to 15 mg/kg body weight, from about 0.005 mg/kg bodyweight to 10 mg/kg body weight.

In some embodiments, the therapeutically effective dose is greater thanabout 0.1 mg/kg body weight, greater than about 0.5 mg/kg body weight,greater than about 1.0 mg/kg body weight, greater than about 3 mg/kgbody weight, greater than about 5 mg/kg body weight, greater than about10 mg/kg body weight, greater than about 15 mg/kg body weight, greaterthan about 20 mg/kg body weight, greater than about 30 mg/kg bodyweight, greater than about 40 mg/kg body weight, greater than about 50mg/kg body weight, greater than about 60 mg/kg body weight, greater thanabout 70 mg/kg body weight, greater than about 80 mg/kg body weight,greater than about 90 mg/kg body weight, greater than about 100 mg/kgbody weight, greater than about 150 mg/kg body weight, greater thanabout 200 mg/kg body weight, greater than about 250 mg/kg body weight,greater than about 300 mg/kg body weight, greater than about 350 mg/kgbody weight, greater than about 400 mg/kg body weight, greater thanabout 450 mg/kg body weight, greater than about 500 mg/kg body weight.In a particular embodiment, the therapeutically effective dose is 1.0mg/kg. In some embodiments, the therapeutically effective dose of 1.0mg/kg is administered intrathecally or intravenously. In someembodiments, the intrathecal dose is or is greater than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 μg. In some embodiments, the intravenous doseis or is greater than 0.5 or 1 mg.

Provided liposomes and compositions may be administered to any desiredtissue. In some embodiments, the FXN mRNA delivered by providedliposomes or compositions is expressed in the tissue in which theliposomes and/or compositions were administered. In some embodiments,the mRNA delivered is expressed in a tissue different from the tissue inwhich the liposomes and/or compositions were administered. Exemplarytissues in which delivered mRNA may be delivered and/or expressedinclude, but are not limited to the liver, kidney, heart, spleen, serum,brain, dorsal root ganglia, skeletal muscle, lymph nodes, skin, and/orcerebrospinal fluid.

According to various embodiments, the timing of expression of deliveredmRNAs can be tuned to suit a particular medical need. In someembodiments, the expression of the protein encoded by delivered mRNA isdetectable 1, 2, 3, 6, 12, 24, 48, 72, 96 hours, 1 week, 2 weeks, or 1month after administration of provided liposomes and/or compositions.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same.

Example 1 Exemplary Liposome Formulations for FXN mRNA Delivery andExpression

This example provides exemplary liposome formulations for effectivedelivery and expression of FXN mRNA in vivo.

Exemplary codon-optimized FXN mRNAs as described above, including SEQ IDNOs: 1, 2, and 4 were synthesized by in vitro transcription from aplasmid DNA template encoding the gene, which was followed by theaddition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G.G.“Recognition of mRNA cap structures by viral and cellular proteins” J.Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail ofapproximately 250 nucleotides in length as determined by gelelectrophoresis. The mRNA encoding frataxin protein also comprised 5′and 3′ untranslated regions (UTRs) (vide supra).

An aqueous-based solution comprising the exemplary mRNA encodingfrataxin protein was combined with one of two ethanol-based lipidsolutions, isolated and dialyzed into the final formulation appropriatefor storage at −80° C.

One lipid solution contained five lipid components to form liposomes.The five biodegradable components all contributed to the final drugproduct characteristics. The first component was a cationic lipid (e.g.,ICE, cKK-E12, CCBene, etc.). Without being bound by theory, thisafforded a positively charged environment at low pH which facilitatesefficient encapsulation of the negatively charged mRNA. It may also playa key role in cell surface interaction to allow for cellular uptake. Thesecond component of the lipid nanoparticle (LNP) was a non-cationiclipid (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)).Without being bound by theory, DOPE is a zwitterionic lipid that hasbeen reported to have fusogenic properties to enhance uptake and releaseof the drug payload. The third component was cholesterol. Without beingbound by theory, cholesterol provided stability giving rigidity to thelipid bilayer. The fourth component was a PEGylated (i.e., PEG-modified)lipid (e.g., 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol(DMG-PEG-2K)). Without being bound by theory, the addition of thisPEGylated lipid provided control over particle size and stability of thenanoparticle and may provide enhanced mucopentrating properties for lunguptake. The fifth component was a sphingomyelin (e.g., brainsphingomyelin). Sphingomyelin in an endogenous lipid component found inall mammalian cells and enriched in central nervous system tissue.Without being bound by theory, the brain sphingomyelin was incorporatedto provide means to represent a more endogenous lipid vesicle for theCNS.

Another lipid solution contained three lipid components to formliposomes. The three components all contributed to the final drugproduct characteristics. The first component was an ionizable lipid(e.g., imidazole cholesterol ester (ICE), cKK-E12, CCBene, etc.).Without being bound by theory, this afforded a positively chargedenvironment at low pH which facilitates efficient encapsulation of thenegatively charged mRNA. It may also play a key role in cell surfaceinteraction to allow for cellular uptake. The second component of theLNP was a non-cationic lipid (e.g.,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)). Without beingbound by theory, DOPE is a zwitterionic lipid that has been reported tohave fusogenic properties to enhance uptake and release of the drugpayload. The third component was a PEGylated (i.e., PEG-modified) lipid(e.g., 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol(DMG-PEG-2K)). Without being bound by theory, the addition of thisPEGylated lipid provided control over particle size and stability of thenanoparticle and may provide enhanced mucopentrating properties for lunguptake. The nominal nitrogen/phosphorus (N/P) charge ratio of the LNPwas 4, and the average particle size range for the mRNA encapsulated inthe LNP was 40-60 nm.

Example 2 In Vitro Transfection of HEK Cells with mRNA-Loaded LiposomeNanoparticles and In Vitro Detection of Produced Frataxin

This example illustrates exemplary methods of transfecting cells withFXN mRNA-loaded liposomes and methods for detecting frataxin protein inthe cells.

Briefly, an aliquot of each selected mRNA was complexed withLipofectamine 2000 and applied to wells containing HEK293 cells fortransfection. Approximately 18 hours later, the cells were harvested andlysed for human FXN analysis.

FXN mRNA was produced as described above in Example 1. Subsequently, inaccordance with the exemplary protocol described above, 4.0, 2.0, 1.0and 0.5 μg of FXN mRNA was transfected into HEK cells. Frataxin proteinwas produced in HEK cells as described above.

HEK cells were extracted with 100 μl or 150 μl of extraction solution(RIPA buffer (Thermo Fisher #89900)+protease inhibitor (Roche1183617001)). Samples were placed on ice for 20 minutes to allow foamsettle, transferred to a new tube, and cleared by centrifuge at 20,000×gfor 10 minutes at 4° C. The supernatant were collected and quantifiedusing a BCA assay. Protein extracts (2.5-30 μg) were mixed with NuPageLDS loading buffer (NP0007), run on 10% bis tris NuPage gels(WG1202BOX), and transferred onto Nitrocellulose membrane (1704159)using the Trans-Blot® Turbo™ Transfer System (Bio-Rad; 1704155). Blotswere imaged and quantified using the LiCor Odyssey imaging system.Frataxin was imaged using mouse anti-human FXN monoclonal antibody(ab110328, Abcam) and GAPDH was imaged using rabbit anti-mouse GAPDH(8884s, Cell Signaling). Conjugated fluorescent secondary antibodieswere used for visualization donkey anti-mouse IgG DαM800CW (LI-COR,926-32210) and goat anti-rabbit IgG GαR680RD (LiCor, 926-68071). Theprotein band fluorescent intensity was measured and quantified using theImage Studio program (LiCor).

Detection of frataxin protein in HEK cells was achieved usingimmunological detection methods (e.g. Western blot). As demonstrated inFIG. 1, the exogenous mature human frataxin protein was detected whencells were transfected with 4.0, 2.0, 1.0 or 0.5 μg of FXN mRNA. Thiswas true for all three tested codon-optimized FXN mRNAs (hFXN-159(comprising SEQ ID NO: 1), hFXN-160 (comprising SEQ ID NO: 2) andhFXN-162 (comprising SEQ ID NO: 4)). Exogenous mature human frataxinprotein was not detected in cells transfected with lipofectamine alone.

Example 3 Intravenous Administration of FXN mRNA-Loaded LiposomeNanoparticles

This example illustrates exemplary methods of administering FXNmRNA-loaded liposomes via intravenous administration that results in invivo expression of frataxin protein and detection of that frataxinprotein in various tissues, including heart tissue, of a subject, andprovides methods for analyzing FXN mRNA and frataxin protein in varioustarget tissues, including heart tissue, of a subject.

All studies were performed using CD-1 mice. Mice were treated witheither human FXN mRNA-loaded cKK-E12-based liposomes (MRT1 comprised SEQID NO: 1, MRT2 comprised SEQ ID NO: 2 and MRT3 comprised SEQ ID NO: 4)or human FXN mRNA-loaded ICE-based liposomes (MRT 4, MRT 5 and MRT 6) bya single bolus tail-vein injection of a 0.5 mg/kg or 1.0 mg/kg dose.Mice were sacrificed 24 hours after dose administration.

Tissues, including liver, heart, spinal cord, dorsal root ganglia (DRG)and brain of each mouse were harvested, and apportioned into separateparts as described herein. For liver collection, 3 medial lobe biopsiesand 2 lateral lobe biopsies were taken, with 1 medial and 1 lateralbiopsy used for testing RNA and 2 medial and 1 lateral biopsy used fortesting protein. For heart collection, the hearts were bisected, withhalf used for testing RNA and half used for testing protein. For spinalcord collection, the spinal cords were divided into thoracic and lumbarportions, with the thoracic portion used for testing RNA and the lumbarportion used for testing protein. For DRG collection, the DRG (target of20 per animal) on each lateral side of T1-L6 were collected. For braincollection, the cerebellum and cerebrum were bisected along the sagittalmidline, with half of each region used for testing RNA and the otherhalf used for testing protein. For each tissue, samples used for RNAtesting were stored in RNALater and samples used for protein testingwere snap frozen in LN₂.

Western Blot Detection of Frataxin Protein in Mouse Tissue Homogenate

Liver or heart or CNS tissues (˜30-100 mg) were transferred to Omni tube(2 mL Tube with 2.8 mm Ceramic Beads) and mixed with 300-700 μL oftissue extraction buffer (RIPA buffer+protease inhibitor) and lysed byusing Omni Bead Ruptor 24 Homogenizer at 5000 rpm for 20 seconds.Samples were placed on ice for 20 minutes to allow foam settle,transferred to a new tube, and cleared by centrifuge at 20,000×g for 10minutes at 4° C. Dorsal Root Ganglia (DRG) lysates were prepared using agrinder due to the small amount of tissue available. 10 to 20 DRG werecollected into a cryo-tube on dry ice and stored at −80° C. Keeping DRGson ice, 50 μL of tissue extraction buffer was added, and DRGs wereground with a motorized pestle (Fisher, pestle_#12-141-364; using amotor #7495410000), incubated on ice for 30-40 minutes, and lysates werecleared by centrifugation at 20,000×g for 10 minutes at 4° C. Thesupernatant was collected and quantified using a BCA assay. Proteinextracts were mixed with NuPage LDS loading buffer (NP0007), run on 10%bis tris NuPage gels (WG1202BOX), and transferred onto Nitrocellulosemembrane (1704159) using the Trans-Blot® Turbo™ Transfer System(Bio-Rad; 1704155). Blots were imaged and quantified using the LiCorOdyssey imaging system. Frataxin was imaged using mouse anti-human FXNmonoclonal antibody (ab110328, Abcam) and GAPDH was imaged using rabbitanti-mouse GAPDH (8884s, Cell Signaling). Conjugated fluorescentsecondary antibodies were used for visualization donkey anti-mouse IgGDαM800CW (LI-COR, 926-32210) and goat anti-rabbit IgG GαR680RD(LiCor,926-68071). The protein band fluorescent intensity was measuredand quantified using the Image Studio program (LiCor).

ELISA Quantification of Frataxin Protein in Mouse Tissue Homogenate

All ELISAs were performed using the commercially available SimpleStepHuman Frataxin ELISA Kit (ab176112). Liver or heart or CNS tissues(˜30-100 mg) were transferred to Omni tube (2 mL Tube with 2.8 mmCeramic Beads) and mix with 300-700 μl of tissue extraction buffer (RIPAbuffer+protease inhibitor) and lysed by using Omni Bead Ruptor 24Homogenizer at 5000 rpm for 20 seconds. Samples were placed on ice for20 minutes to allow foam settle, transferred to a new tube, and clearedby centrifuge at 20,000×g for 10 minutes at 4° C. The supernatant wascollected and quantified using a BCA assay. The extracts were dilutedwith 1× Cell Extraction buffer to fit within the linear range of therecombinant hFXN standard, starting at roughly 20 ng/μL. 50 μL of eachextract was transferred to a well in 8-well strips in a 96-well plateformat and mixed with 50 μL of the Antibody Cocktail. Plates were sealedand incubated at room temp for 70 minutes on a plate shaker set to 400rpm. Plates were washed 3×350 μL with 1× Wash Buffer PT. Buffer wascompletely removed by aspiration at each step. The plate was invertedand blotted on clean paper towels to remove excess liquid. 100 μL of TMBSubstrate was added to each well and incubated for 10 minutes in thedark on a plate shaker set to 400 rpm. 100 μL of Stop Solution was addedto each well, the plate was shaken for 1 minute to mix, and the OD wasrecorded at 450 nm. Background was subtracted for each well. Readingsfrom the recombinant hFXN standard (0-800 pg/mL) were used to constructa standard curve. A log/log four parameter algorithm (4PL) was used tofit the standard curve and the protein concentrations of the unknownsamples were interpolated from that curve.

Results

Detection of frataxin protein in the livers of the treated mice wasachieved using antibody-based methods (e.g. Western blot). Asdemonstrated in FIG. 2, the exogenous human frataxin protein wasdetected in all three mRNA-liposome combinations (MRT1 comprised SEQ IDNO: 1, MRT2 comprised SEQ ID NO: 2 and MRT3 comprised SEQ ID NO: 4).However, there was differential expression of the intermediate humanfrataxin protein observed between the three mRNA treatments.

Additionally, the mRNA-derived human frataxin protein was detected as arobust signal with ELISA performed on liver samples. As shown in FIG.3A, both 0.5 mg/kg and 1.0 mg/kg doses of mRNA delivered via IVadministration resulted in human frataxin protein being delivered to anddetectable in the liver, as compared to controls. FIG. 3B shows that arobust mRNA signal was detected by RT qPCR from liver samples.

The mRNA-derived human frataxin protein also was detected with ELISA inthe heart tissue and in the DRG of subjects. As shown in FIG. 4A, both0.5 mg/kg and 1.0 mg/kg doses of mRNA delivered via IV administrationresulted in human frataxin protein being delivered to and detectable inthe heart, as compared to controls. As shown in FIG. 4B, both 0.5 mg/kgand 1.0 mg/kg doses of mRNA delivered via IV administration resulted inhuman frataxin protein being delivered to and detectable in the DRG, ascompared to controls.

Example 4 Intrathecal Administration of FXN mRNA-Loaded LiposomeNanoparticles

This example illustrates exemplary methods of administering FXNmRNA-loaded liposomes via intrathecal administration that results in invivo expression of frataxin protein and detection of that frataxinprotein in various tissues, including cerebellum tissue, of a subject,and provides methods for analyzing FXN mRNA and frataxin protein invarious target tissues, including cerebellum tissue, of a subject.

All studies were performed using CD-1 mice. Mice were treated witheither human FXN mRNA-loaded cKK-E12-based liposomes (MRT 1, MRT 2 andMRT 3) or human FXN mRNA-loaded ICE-based liposomes (MRT 4, MRT 5 andMRT 6) by a single intrathecal (IT) bolus of a 5 μg/animal, 10 μg/animalor 20 μg/animal dose while under inhaled isoflurane anesthesia. Micewere sacrificed 24 hours after dose administration.

Tissues, including liver, spinal cord, dorsal root ganglia (DRG) andbrain, including cerebellum, of each mouse were harvested, andapportioned into separate parts as described herein. For livercollection, 3 medial lobe biopsies and 2 lateral lobe biopsies weretaken, with 1 medial and 1 lateral biopsy used for testing RNA and 2medial and 1 lateral biopsy used for testing protein. For spinal cordcollection, the spinal cords were divided into thoracic and lumbarportions, with the thoracic portion used for testing RNA and the lumbarportion used for testing protein. For DRG collection, the DRG (target of20 per animal) on each lateral side of T1-L6 were collected. For braincollection, the cerebellum and cerebrum were bisected along the sagittalmidline, with half of each region used for testing RNA and the otherhalf used for testing protein. For each tissue, samples used for RNAtesting were stored in RNALater and samples used for protein testingwere snap frozen in LN₂.

Western Blot Detection of Frataxin Protein in Mouse Tissue Homogenate

Liver or heart or CNS tissues (˜30-100 mg) were transferred to Omni tube(2 mL Tube with 2.8 mm Ceramic Beads) and mixed with 300-700 μL oftissue extraction buffer (RIPA buffer+protease inhibitor) and lysed byusing Omni Bead Ruptor 24 Homogenizer at 5000 rpm for 20 seconds.Samples were placed on ice for 20 minutes to allow foam settle,transferred to a new tube, and cleared by centrifuge at 20,000×g for 10minutes at 4° C. Dorsal Root Ganglia (DRG) lysates were prepared using agrinder due to the small amount of tissue available. 10 to 20 DRG werecollected into a cryo-tube on dry ice and stored at −80° C. Keeping DRGson ice, 50 μL of tissue extraction buffer was added, and DRGs wereground with a motorized pestle (Fisher, pestle_#12-141-364; using amotor #7495410000), incubated on ice for 30-40 minutes, and lysates werecleared by centrifugation at 20,000×g for 10 minutes at 4° C. Thesupernatant was collected and quantified using a BCA assay. Proteinextracts were mixed with NuPage LDS loading buffer (NP0007), run on 10%bis tris NuPage gels (WG1202BOX), and transferred onto Nitrocellulosemembrane (1704159) using the Trans-Blot® Turbo™ Transfer System(Bio-Rad; 1704155). Blots were imaged and quantified using the LiCorOdyssey imaging system. Frataxin was imaged using mouse anti-human FXNmonoclonal antibody (ab110328, Abcam) and GAPDH was imaged using rabbitanti-mouse GAPDH (8884s, Cell Signaling). Conjugated fluorescentsecondary antibodies were used for visualization donkey anti-mouse IgGDαM800CW (LI-COR, 926-32210) and goat anti-rabbit IgG GαR680RD(LiCor,926-68071). The protein band fluorescent intensity was measuredand quantified using the Image Studio program (LiCor).

ELISA Quantification of Frataxin Protein in Mouse Tissue Homogenate

All ELISAs were performed using the commercially available SimpleStepHuman Frataxin ELISA Kit (ab176112). Liver or heart or CNS tissues(˜30-100 mg) were transferred to Omni tube (2 mL Tube with 2.8 mmCeramic Beads) and mix with 300-700 μl of tissue extraction buffer (RIPAbuffer+protease inhibitor) and lysed by using Omni Bead Ruptor 24Homogenizer at 5000 rpm for 20 seconds. Samples were placed on ice for20 minutes to allow foam settle, transferred to a new tube, and clearedby centrifuge at 20,000×g for 10 minutes at 4° C. The supernatant wascollected and quantified using a BCA assay. The extracts were dilutedwith 1× Cell Extraction buffer to fit within the linear range of therecombinant hFXN standard, starting at roughly 20 ng/μL. 50 μL of eachextract was transferred to a well in 8-well strips in a 96-well plateformat and mixed with 50 μL of the Antibody Cocktail. Plates were sealedand incubated at room temp for 70 minutes on a plate shaker set to 400rpm. Plates were washed 3×350 μL with 1× Wash Buffer PT. Buffer wascompletely removed by aspiration at each step. The plate was invertedand blotted on clean paper towels to remove excess liquid. 100 μL of TMBSubstrate was added to each well and incubated for 10 minutes in thedark on a plate shaker set to 400 rpm. 100 μL of Stop Solution was addedto each well, the plate was shaken for 1 minute to mix, and the OD wasrecorded at 450 nm. Background was subtracted for each well. Readingsfrom the recombinant hFXN standard (0-800 pg/mL) were used to constructa standard curve. A log/log four parameter algorithm (4PL) was used tofit the standard curve and the protein concentrations of the unknownsamples were interpolated from that curve.

Results

Detection of frataxin protein in the tissues of the treated mice wasachieved using antibody-based methods (e.g. Western blot). Asdemonstrated in FIG. 5A, the exogenous human frataxin protein wasdetected in all three mRNA-liposome combinations (MRT1 comprised SEQ IDNO: 1, MRT2 comprised SEQ ID NO: 2 and MRT comprised SEQ ID NO: 4) inDRG samples when animals were treated with 20 μg of hFXN mRNA. There wassome variation, but robust expression was observed in most samples.Similar results were observed in spinal cord samples (FIG. 5B).

Exogenous human frataxin protein was also detected in liver samples fromanimals treated with 20 μg of hFXN mRNA. As demonstrated in FIG. 6,robust levels of mature human frataxin protein was observed by Westernblot, though there was differential expression of the intermediate humanfrataxin protein observed between the three mRNA treatments, similar toobserved results from the IV administration study. Robust human frataxinexpression was also detected by ELISA in liver samples from animalstreated with all three doses (5 μg, 10 μg and 20 μg) of hFXN mRNA (FIG.7).

Additionally, the mRNA-derived human frataxin protein was detected as arobust signal with ELISA performed on DRG and spinal cord samples. Asshown in FIG. 8A, the 5 μg, 10 μg and 20 μg doses of mRNA delivered viaIT administration resulted in human frataxin protein being delivered toand detectable in the DRG, as compared to controls. The same doses ofmRNA also lead to increased protein expression in the spinal cord, ascompared to controls (FIG. 8B).

Finally, when ELISA was performed on samples from the cerebellum andcerebrum of treated mice (FIG. 9A and FIG. 9B, respectively), the 5 μg,10 μg and 20 μg doses of mRNA delivered via IT administration resultedin human frataxin protein being delivered to and detectable in thecerebellum, as compared to controls.

Example 5 Duration of Action of FXN mRNA-Loaded Liposome NanoparticlesIn Vivo

This example illustrates that administering FXN mRNA-loaded liposomesvia intrathecal administration results in sustained in vivo expressionof frataxin protein in various tissues important for therapeuticefficacy.

All studies were performed using CD-1 mice. Mice were treated with humanFXN mRNA-loaded ICE-based liposomes (MRT4) by a single intrathecal (IT)bolus of a 2 μg/animal or 20 μg/animal dose while under inhaledisofluorane anesthesia. Mice were treated on Day 1 and sacrificed ateither Day 4, Day 8, Day 15, or Day 29 (i.e., 3 days, 7 days, 14 days,or 28 days after dose administration, respectfully).

Tissues, including dorsal root ganglia (DRG), and spinal cord of eachmouse were harvested, and apportioned into separate parts as describedherein. For spinal cord collection, the spinal cords were divided intothoracic and lumbar portions, with the thoracic portion used for testingRNA and the lumbar portion used for testing protein. For DRG collection,the DRG (target of 20 per animal) on each lateral side of T1-L6 werecollected. For brain collection, the cerebrum was bisected along thesagittal midline, with half of each region used for testing RNA and theother half for testing protein. For each tissue, both halves used forRNA and protein were snap frozen in LN₂.

ELISA Quantification of Frataxin Protein in Mouse Tissue Homogenate

All ELISAs were performed using the commercially available SimpleStepHuman Frataxin ELISA Kit (ab176112). Liver or heart or CNS tissues(˜30-100 mg) were transferred to Omni tube (2 mL Tube with 2.8 mmCeramic Beads) and mix with 300-700 μl of tissue extraction buffer (RIPAbuffer+protease inhibitor) and lysed by using Omni Bead Ruptor 24Homogenizer at 5000 rpm for 20 seconds. Samples were placed on ice for20 minutes to allow foam settle, transferred to a new tube, and clearedby centrifuge at 20,000×g for 10 minutes at 4° C. The supernatant wascollected and quantified using a BCA assay. The extracts were dilutedwith 1× Cell Extraction buffer to fit within the linear range of therecombinant hFXN standard, starting at roughly 20 ng/μL. 50 μL of eachextract was transferred to a well in 8-well strips in a 96-well plateformat and mixed with 50 μL of the Antibody Cocktail. Plates were sealedand incubated at room temp for 70 minutes on a plate shaker set to 400rpm. Plates were washed 3×350 μL with 1× Wash Buffer PT. Buffer wascompletely removed by aspiration at each step. The plate was invertedand blotted on clean paper towels to remove excess liquid. 100 μL of TMBSubstrate was added to each well and incubated for 10 minutes in thedark on a plate shaker set to 400 rpm. 100 μL of Stop Solution was addedto each well, the plate was shaken for 1 minute to mix, and the OD wasrecorded at 450 nm. Background was subtracted for each well. Readingsfrom the recombinant hFXN standard (0-800 pg/mL) were used to constructa standard curve. A log/log four parameter algorithm (4PL) was used tofit the standard curve and the protein concentrations of the unknownsamples were interpolated from that curve.

Results

Detection of FXN protein in the DRGs of treated mice was achieved usingan antibody-based method (i.e. ELISA). As demonstrated in FIG. 10A, theexogenous FXN protein was detected in animals at 2μg/animal and 20μg/animal (MRT 4) for at least 14 days following administration. Thebaseline levels of human FXN levels in untreated mice is depicted as adashed line in FIG. 10A.

Additionally, the mRNA-derived human frataxin protein was detected inthe spinal cord with ELISA. AS shown in FIG. 10B, the 2 μg/animal and 20μg/animal doses of mRNA delivered via IT administration resulted inhuman frataxin protein being delivered and detectable in the spinalcord, as compared to untreated controls (dashed line in FIG. 10B).

These results demonstrate that delivery of FXN mRNA-loaded liposomesaccording to the present invention results in sustained in vivoexpression of frataxin protein in those tissues important for treatmentof treating Friedreich's ataxia.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

We claim:
 1. A method of treating Friedreich's ataxia (FRDA), comprisingadministering to a subject in need of treatment a composition comprisingan mRNA encoding a frataxin protein, wherein the composition isadministered via intrathecal (IT) delivery, wherein the mRNA comprisessequence identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4, and wherein the administering of the composition results inexpression of the frataxin protein detectable in the cerebellum of thesubject.
 2. The method of claim 1, wherein the administering of thecomposition results in expression of the frataxin protein detectable inthe liver, the spinal cord, the brain, and/or at least one dorsal rootganglion (DRG) of the subject.
 3. The method of claim 1, wherein themRNA is encapsulated within a liposome.
 4. The method of claim 3,wherein the liposome comprises one or more cationic lipids, one or morenon-cationic lipids, and one or more PEG-modified lipids.
 5. The methodof claim 4, wherein the one or more cationic lipids comprise a cationiclipid selected from the group consisting of C12-200, MC3, DLinDMA,DLinkC2DMA, cKK-E12, ICE (Imidazole-based), HGT5000, HGT5001 (CCBene),OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC,DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP,DLincarbDAP, DLinCDAP, DLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, andcombinations thereof.
 6. The method of claim 4, wherein the liposomefurther comprises a sphingomyelin.
 7. The method of claim 1, wherein themRNA is administered once a week, once a month, twice a month, or onceevery 14 days.
 8. A method of delivering frataxin in vivo, comprisingadministering periodically to a subject with Friedreich's ataxia (FRDA)a composition comprising an mRNA encoding a frataxin protein whereinthat the frataxin protein is expressed in vivo at a level of at least10% of a normal control level in cerebellum of the subject, wherein thecomposition is administered to via intrathecal (IT) delivery, andwherein the mRNA comprises sequence identical to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, or SEQ ID NO:
 4. 9. The method of claim 8, whereinthe frataxin protein is expressed in the liver, the spinal cord, thebrain, and/or at least one dorsal root ganglion (DRG) of the subject.10. The method of claim 8, wherein the mRNA is administered once a week,once a month, twice a month, or once every 14 days.